Fatty acid desaturases from primula

ABSTRACT

The invention relates generally to methods and compositions concerning desaturase enzymes that modulate the number and location of double bonds in long chain poly-unsaturated fatty acids (LC-PUFA&#39;s). In particular, the invention relates to methods and compositions for improving omega-3 fatty acid profiles in plant products and parts using desaturase enzymes and nucleic acids encoding for such enzymes. In particular embodiments, the desaturase enzymes are  Primula  Δ6-desaturases. Also provided are improved soybean oil compositions having SDA and a beneficial overall content of omega-3 fatty acids relative to omega-6 fatty acids.

This application claims the priority of U.S. Provisional PatentApplication Ser. No. 60/496,751, filed Aug. 21, 2003, the entiredisclosure of which is specifically incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to desaturase enzymes that modulate thenumber and location of double bonds in long chain poly-unsaturated fattyacids (LC-PUFA's). In particular, the invention relates to improvementof fatty acid profiles using desaturase enzymes and nucleic acidsencoding such desaturase enzymes.

2. Description of the Related Art

The primary products of fatty acid biosynthesis in most organisms are16- and 18-carbon compounds. The relative ratio of chain lengths anddegree of unsaturation of these fatty acids vary widely among species.Mammals, for example, produce primarily saturated and monounsaturatedfatty acids, while most higher plants produce fatty acids with one, two,or three double bonds, the latter two comprising polyunsaturated fattyacids (PUFA's).

Two main families of PUFAs are the omega-3 fatty acids (also representedas “n−3” fatty acids), exemplified by eicosapentaenoic acid (EPA, 20:4,n−3), and the omega-6 fatty acids (also represented as “n−6” fattyacids), exemplified by arachidonic acid (ARA, 20:4, n−6). PUFAs areimportant components of the plasma membrane of the cell and adiposetissue, where they may be found in such forms as phospholipids and astriglycerides, respectively. PUFAs are necessary for proper developmentin mammals, particularly in the developing infant brain, and for tissueformation and repair.

Several disorders respond to treatment with fatty acids. Supplementationwith PUFAs has been shown to reduce the rate of restenosis afterangioplasty. The health benefits of certain dietary omega-3 fatty acidsfor cardiovascular disease and rheumatoid arthritis also have been welldocumented (Simopoulos, 1997; James et al., 2000). Further, PUFAs havebeen suggested for use in treatments for asthma and psoriasis. Evidenceindicates that PUFAs may be involved in calcium metabolism, suggestingthat PUFAs may be useful in the treatment or prevention of osteoporosisand of kidney or urinary tract stones. The majority of evidence forhealth benefits applies to the long chain omega-3 fats, EPA anddocosahexanenoic acid (DHA, 22:6) which are in fish and fish oil. Withthis base of evidence, health authorities and nutritionists in Canada(Scientific Review Committee, 1990, Nutrition Recommendations, Ministerof National Health and Welfare, Canada, Ottowa), Europe (de Deckerer etal., 1998), the United Kingdom (The British Nutrition Foundation, 1992,Unsaturated fatty-acids—nutritional and physiological significance: Thereport of the British Nutrition Foundation's Task Force, Chapman andHall, London), and the United States (Simopoulos et al., 1999) haverecommended increased dietary consumption of these PUFAs.

PUFAs also can be used to treat diabetes (U.S. Pat. No. 4,826,877;Horrobin et al., 1993). Altered fatty acid metabolism and compositionhave been demonstrated in diabetic animals. These alterations have beensuggested to be involved in some of the long-term complicationsresulting from diabetes, including retinopathy, neuropathy, nephropathyand reproductive system damage. Primrose oil, which contains γ-linolenicacid (GLA, 18:3, Δ6, 9, 12), has been shown to prevent and reversediabetic nerve damage.

PUFAs, such as linoleic acid (LA, 18:2, Δ9, 12) and α-linolenic acid(ALA, 18:3, Δ9, 12, 15), are regarded as essential fatty acids in thediet because mammals lack the ability to synthesize these acids.However, when ingested, mammals have the ability to metabolize LA andALA to form the n−6 and n−3 families of long-chain polyunsaturated fattyacids (LC-PUFA). These LC-PUFA's are important cellular componentsconferring fluidity to membranes and functioning as precursors ofbiologically active eicosanoids such as prostaglandins, prostacyclins,and leukotrienes, which regulate normal physiological functions.Arachidonic acid is the principal precursor for the synthesis ofeicosanoids, which include leukotrienes, prostaglandins, andthromboxanes, and which also play a role in the inflammation process.Administration of an omega-3 fatty acid, such as SDA, has been shown toinhibit biosynthesis of leukotrienes (U.S. Pat. No. 5,158,975). Theconsumption of SDA has been shown to lead to a decrease in blood levelsof proinflammatory cytokines TNF-α and IL-1β (PCT US 0306870).

In mammals, the formation of LC-PUFA is rate-limited by the step of Δ6desaturation, which converts LA to γ-linolenic acid (GLA, 18:3, Δ6, 9,12) and ALA to SDA (18:4, Δ6, 9, 12, 15). Many physiological andpathological conditions have been shown to depress this metabolic stepeven further, and consequently, the production of LC-PUFA. To overcomethe rate-limiting step and increase tissue levels of EPA, one couldconsume large amounts of ALA. However, consumption of just moderateamounts of SDA provides an efficient source of EPA, as SDA is about fourtimes more efficient than ALA at elevating tissue EPA levels in humans(copending U.S. application Ser. No. 10/384,369). In the same studies,SDA administration was also able to increase the tissue levels ofdocosapentaenoic acid (DPA), which is an elongation product of EPA.Alternatively, bypassing the Δ6-desaturation via dietary supplementationwith EPA or DHA can effectively alleviate many pathological diseasesassociated with low levels of PUFA. However, as set forth in more detailbelow, currently available sources of PUFA are not desirable for amultitude of reasons. The need for a reliable and economical source ofPUFA's has spurred interest in alternative sources of PUFA's.

Major long chain PUFAs of importance include DHA and EPA, which areprimarily found in different types of fish oil, and ARA, found infilamentous fungi such as Mortierella. For DHA, a number of sourcesexist for commercial production including a variety of marine organisms,oils obtained from cold water marine fish, and egg yolk fractions.Commercial sources of SDA include the plant genera Trichodesma, Borago(borage) and Echium. However, there are several disadvantages associatedwith commercial production of PUFAs from natural sources. Naturalsources of PUFAs, such as animals and plants, tend to have highlyheterogeneous oil compositions. The oils obtained from these sourcestherefore can require extensive purification to separate out one or moredesired PUFAs or to produce an oil which is enriched in one or morePUFAs.

Natural sources of PUFAs also are subject to uncontrollable fluctuationsin availability. Fish stocks may undergo natural variation or may bedepleted by overfishing. In addition, even with overwhelming evidence oftheir therapeutic benefits, dietary recommendations regarding omega-3fatty acids are not heeded. Fish oils have unpleasant tastes and odors,which may be impossible to economically separate from the desiredproduct, and can render such products unacceptable as food supplements.Animal oils, and particularly fish oils, can accumulate environmentalpollutants. Foods may be enriched with fish oils, but again, suchenrichment is problematic because of cost and declining fish stocksworldwide. This problem is also an impediment to consumption and intakeof whole fish. Nonetheless, if the health messages to increase fishintake were embraced by communities, there would likely be a problem inmeeting demand for fish. Furthermore, there are problems withsustainability of this industry, which relies heavily on wild fishstocks for aquaculture feed (Naylor et al., 2000).

Other natural limitations favor a novel approach for the production ofomega-3 fatty acids. Weather and disease can cause fluctuation in yieldsfrom both fish and plant sources. Cropland available for production ofalternate oil-producing crops is subject to competition from the steadyexpansion of human populations and the associated increased need forfood production on the remaining arable land. Crops that do producePUFAs, such as borage, have not been adapted to commercial growth andmay not perform well in monoculture. Growth of such crops is thus noteconomically competitive where more profitable and better-establishedcrops can be grown. Large scale fermentation of organisms such asMortierella is also expensive. Natural animal tissues contain lowamounts of ARA and are difficult to process. Microorganisms such asPorphyridium and Mortierella are difficult to cultivate on a commercialscale.

A number of enzymes are involved in the biosynthesis of PUFAs. LA (18:2,Δ9, 12) is produced from oleic acid (OA, 18:1, Δ9) by a Δ12-desaturasewhile ALA (18:3, Δ9, 12, 15) is produced from LA by a Δ15-desaturase.SDA (18:4, Δ6, 9, 12, 15) and GLA (18:3, Δ6, 9, 12) are produced from LAand ALA by a Δ6-desaturase. However, as stated above, mammals cannotdesaturate beyond the Δ9 position and therefore cannot convert oleicacid into LA. Likewise, ALA cannot be synthesized by mammals. Othereukaryotes, including fungi and plants, have enzymes which desaturate atthe carbon 12 and carbon 15 position. The major polyunsaturated fattyacids of animals therefore are derived from diet via the subsequentdesaturation and elongation of dietary LA and ALA.

Various genes encoding desaturases have been described. For example,U.S. Pat. No. 5,952,544 describes nucleic acid fragments isolated andcloned from Brassica napus that encode fatty acid desaturase enzymes.Expression of the nucleic acid fragments of the '544 patent resulted inaccumulation of ALA. However, in transgenic plants expressing the B.napus Δ15-desaturase, substantial LA remains unconverted by thedesaturase. More active enzymes that convert greater amounts of LA toALA would be advantageous. Increased ALA levels allow a Δ6-desaturase,when co-expressed with a nucleic acid encoding for the Δ15-desaturase,to act upon the ALA, thereby producing greater levels of SDA. Because ofthe multitude of beneficial uses for SDA, there is a need to create asubstantial increase in the yield of SDA.

Nucleic acids from a number of sources have been sought for use inincreasing SDA yield. However, innovations that would allow for improvedcommercial production in land-based crops are still needed (see, e.g.,Reed et al., 2000). Furthermore, the use of desaturase polynucleotidesderived from organisms such as Caenorhabditis elegans (Meesapyodsuk etal., 2000) is not ideal for the commercial production of enriched plantseed oils. Genes encoding Δ6-desaturases have been isolated from twospecies of Primula, P. farinosa and P. vialii, and these found to beactive in yeast, but the function in plants was not shown (Sayanova etal., 2003).

Therefore, it would be advantageous to obtain genetic material involvedin PUFA biosynthesis and to express the isolated material in a plantsystem, in particular, a land-based terrestrial crop plant system, whichcan be manipulated to provide production of commercial quantities of oneor more PUFA's. There is also a need to increase omega-3 fat intake inhumans and animals. Thus there is a need to provide a wide range ofomega-3 enriched foods and food supplements so that subjects can choosefeed, feed ingredients, food and food ingredients which suit their usualdietary habits. Particularly advantageous would be seed oils withincreased SDA.

Currently there is only one omega-3 fatty acid, ALA, available invegetable oils. However, there is poor conversion of ingested ALA to thelonger-chain omega-3 fatty acids such as EPA and DHA. It has beendemonstrated in copending U.S. application Ser. No. 10/384,369 for“Treatment And Prevention Of Inflammatory Disorders,” that elevating ALAintake from the community average of 1/g day to 14 g/day by use offlaxseed oil only modestly increased plasma phospholipid EPA levels. A14-fold increase in ALA intake resulted in a 2-fold increase in plasmaphospholipid EPA (Manzioris et al., 1994). Thus, to that end, there is aneed for efficient and commercially viable production of PUFAs usingfatty acid desaturases, genes encoding them, and recombinant methods ofproducing them. A need also exists for oils containing higher relativeproportions of specific PUFAs, and food compositions and supplementscontaining them. A need also exists for reliable economical methods ofproducing specific PUFA's.

Despite inefficiencies and low yields as described above, the productionof omega-3 fatty acids via the terrestrial food chain is an enterprisebeneficial to public health and, in particular, the production of SDA.SDA is important because, as described above, there is low conversion ofALA to EPA. This is because the initial enzyme in the conversion,Δ6-desaturase, has low activity in humans and is rate-limiting. Evidencethat Δ6-desaturase is rate-limiting is provided by studies whichdemonstrate that the conversion of its substrate, ALA, is less efficientthan the conversion of its product, SDA to EPA in mice and rats(Yamazaki et al., 1992; Huang, 1991).

Based on such studies, it is seen that in commercial oilseed crops, suchas canola, soybean, corn, sunflower, safflower, or flax, the conversionof some fraction of the mono and polyunsaturated fatty acids that typifytheir seed oil to SDA requires the seed-specific expression of multipledesaturase enzymes, that includes Δ6-, Δ12- and/or Δ15-desaturases. Oilsderived from plants expressing elevated levels of Δ6, Δ12, andΔ15-desaturases are rich in SDA and other omega-3 fatty acids. Such oilscan be utilized to produce foods and food supplements enriched inomega-3 fatty acids and consumption of such foods effectively increasestissue levels of EPA and DHA. Foods and foodstuffs, such as milk,margarine and sausages, all made or prepared with omega-3 enriched oils,will result in therapeutic benefits. It has been shown that subjects canhave an omega-3 intake comparable to EPA and DHA of at least 1.8 g/daywithout altering their dietary habits by utilizing foods containing oilsenriched with omega-3 fatty acids. Thus, there exists a strong need fornovel nucleic acids of Δ6-desaturases for use in transgenic crop plantswith oils enriched in PUFAs, as well as the improved oils producedthereby.

SUMMARY OF THE INVENTION

In one aspect, the invention provides isolated nucleic acids encoding apolypeptide capable of desaturating a fatty acid molecule at carbon 6(Δ6-desaturase). These may be used to transform cells or modify thefatty acid composition of a plant or the oil produced by a plant. Oneembodiment of the invention is an isolated polynucleotide sequenceisolated from a Primula species having unique desaturase activity. Incertain embodiments, the isolated polynucleotides are isolated, forexample, from Primula juliae, P. alpicola, P. waltonii, P. farinosa orP. florindae. In certain further embodiments of the invention, thepolynucleotides encode a polypeptide having at least 90% sequenceidentity to the polypeptide sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:46 or SEQ ID NO:48,including at least about 92%, 95%, 98% and 99% homology to thesesequences. Those of skill in the art will recognize that, as thesesequences are related, a given polypeptide may simultaneously share 90%or greater homology to more than one of these polypeptide sequences. Incertain embodiments, a sequence provided by the invention has asubstrate selectivity for >linolenic acid relative to linoleic acid, asdescribed herein. In further embodiments, there is at least 2:1substrate selectivity for α-linolenic acid relative to linoleic acid,including from about 2:1 to about 2.9:1.

In another aspect, the invention provides an isolated polynucleotidethat encodes a polypeptide having desaturase activity that desaturates afatty acid molecule at carbon 6, comprising a sequence selected from thegroup consisting of: (a) a polynucleotide encoding the polypeptide ofSEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQID NO:46 or SEQ ID NO:48; (b) a polynucleotide comprising the nucleicacid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:45 or SEQ ID NO:47; (c) a polynucleotidehybridizing to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:21, SEQ ID NO:23, SEQID NO:25, SEQ ID NO:45 or SEQ ID NO:47, or a complement thereof, underconditions of 5×SSC, 50% formamide and 42° C.; and (d) a polynucleotideencoding a polypeptide with at least 90% sequence identity to apolypeptide sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:46 or SEQ ID NO:48.

In yet another aspect, the invention provides a recombinant vectorcomprising an isolated polynucleotide in accordance with the invention.The term “recombinant vector” as used herein, includes any recombinantsegment of DNA that one desires to introduce into a host cell, tissueand/or organism, and specifically includes expression cassettes isolatedfrom a starting polynucleotide. A recombinant vector may be linear orcircular. In various aspects, a recombinant vector may comprise at leastone additional sequence chosen from the group consisting of: regulatorysequences operatively coupled to the polynucleotide; selection markersoperatively coupled to the polynucleotide; marker sequences operativelycoupled to the polynucleotide; a purification moiety operatively coupledto the polynucleotide; and a targeting sequence operatively coupled tothe polynucleotide.

In still yet another aspect, the invention provides cells, such asmammal, plant, insect, yeast and bacteria cells transformed with thepolynucleotides of the instant invention. In a further embodiment, thecells are transformed with recombinant vectors containing constitutiveand tissue-specific promoters in addition to the polynucleotides of theinstant invention. In certain embodiments of the invention, such cellsmay be further defined as transformed with a nucleic acid sequenceencoding a polypeptide having desaturase activity that desaturates afatty acid molecule at carbon 12 and/or 15.

The invention also provides a polypeptide comprising the amino acidsequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:46 or SEQ ID NO:48; or a fragment thereof havingdesaturase activity that desaturates a fatty acid molecule at carbon 6.

Still yet another aspect of the invention provides a method of producingseed oil containing omega-3 fatty acids from plant seeds, comprising thesteps of (a) obtaining seeds of a plant according to the invention; and(b) extracting the oil from said seeds. Examples of such a plant includecanola, soy, soybeans, rapeseed, sunflower, cotton, cocoa, peanut,safflower, coconut, flax, oil palm, oilseed Brassica napus, and corn.Preferred methods of transforming such plant cells include the use of Tiand Ri plasmids of Agrobacterium, electroporation, and high-velocityballistic bombardment.

In still yet another aspect, the invention provides a method ofproducing a plant comprising seed oil containing altered levels ofomega-3 fatty acids comprising introducing a recombinant vector of theinvention into an oil-producing plant. In the method, introducing therecombinant vector may comprise genetic transformation. In embodiment,transformation comprises the steps of: (a) transforming a plant cellwith a recombinant vector of the invention; and (b) regenerating theplant from the plant cell, wherein the plant has altered levels ofomega-3 fatty acids relative to a corresponding plant of the samegenotype that was not transformed with the vector. In the method, theplant may, for example, be selected from the group consisting ofArabidopsis thaliana, oilseed Brassica, rapeseed, sunflower, safflower,canola, corn, soybean, cotton, flax, jojoba, Chinese tallow tree,tobacco, cocoa, peanut, fruit plants, citrus plants, and plantsproducing nuts and berries. The plant may be further defined astransformed with a nucleic acid sequence encoding a polypeptide havingdesaturase activity that desaturates a fatty acid molecule at carbon 12and/or 15. The plant may comprise increased SDA. The method may furthercomprise introducing the recombinant vector into a plurality ofoil-producing plants and screening the plants or progeny thereof havinginherited the recombinant vector for a plant having a desired profile ofomega-3 fatty acids.

In still yet another aspect, the invention provides an endogenoussoybean seed oil having a SDA content of from about 5% to about 50% anda gamma-linoleic acid content of less than about 10%. The SDA contentmay, in certain embodiments, be further defined as from about 5% toabout 32%, from about 5% to about 35%, from about 15% to about 30%, fromabout 22% to about 30%, and from about 22% to about 40%. Thegamma-linoleic acid content may, in further embodiments, be defined asless than about 10, 8, 5 and/or about 3%. In particular embodiments, thestearidonic acid content may be from about 15% to about 35% and thegamma-linoleic acid content less than 5%. In still further embodiments,the seed may comprise a ratio of omega-3 to omega-6 fatty acids of fromabout 0.35:1 to about 3.5:1, including from about 1:1 to about 3.5:1 andfrom about 2:1 to about 3.5:1.

In still yet another aspect, the invention provides a method ofincreasing the nutritional value of an edible product for human oranimal consumption, comprising adding a soybean seed oil provided by theinvention to the edible product. In certain embodiments, the product ishuman and/or animal food. The edible product may also be animal feedand/or a food supplement. In the method, the soybean seed oil mayincrease the SDA content of the edible product and/or may increase theratio of omega-3 to omega-6 fatty acids of the edible product. Theedible product may lack SDA prior to adding the soybean seed oil.

In still yet another aspect, the invention provides a method ofmanufacturing food or feed, comprising adding a soybean seed oilprovided by the invention to starting food or feed ingredients toproduce the food or feed. In certain embodiments, the method is furtherdefined as a method of manufacturing food and/or feed. The inventionalso provides food or feed made by the method.

In still yet another aspect, the invention comprises a method ofproviding SDA to a human or animal, comprising administering the soybeanseed oil of claim 1 to said human or animal. In the method, the soybeanseed oil may be administered in an edible composition, including food orfeed. Examples of food include beverages, infused foods, sauces,condiments, salad dressings, fruit juices, syrups, desserts, icings andfillings, soft frozen products, confections or intermediate food. Theedible composition may be substantially a liquid or solid. The ediblecomposition may also be a food supplement and/or nutraceutical. In themethod, the soybean seed oil may be administered to a human and/or ananimal. Examples of animals the oil may be administered to includelivestock or poultry.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows alignment of Primula juliae Δ6 desaturases PjD6D-1 andPjD6D-2 (SEQ ID NOs:4 and 5), Primula alpicola Pa6D-1 and Pa6D-2 (SEQ IDNOs: 22 and 24), Primula waltonii PwD6D (SEQ ID NO:26), Primula farinosaD6D-2 (SEQ ID NO:46), Primula florindae D6D (SEQ ID NO:48), Boragooficinalis D6D (SEQ ID NO:59) and Echium gentianoides D6D (SEQ IDNO:60).

FIG. 2 shows map of vector pMON67011.

FIG. 3 shows map of vector pMON83950.

FIG. 4 shows map of vector pMON77245.

FIG. 5 shows map of vector pMON77247.

FIG. 6 shows map of vector pMON82821.

FIG. 7 shows map of vector pMON82822.

FIG. 8 shows map of vector pMON83961.

FIG. 9 shows map of vector pMON83962.

FIG. 10 shows map of vector pMON83963.

FIG. 11 shows map of vector pMON83964.

FIG. 12 shows map of vector pMON83965.

FIG. 13 shows map of vector pMON83966.

DETAILED DESCRIPTION OF THE INVENTION

The invention overcomes the limitations of the prior art by providingmethods and compositions for creation of plants with improved PUFAcontent. The modification of fatty acid content of an organism such as aplant presents many advantages, including improved nutrition and healthbenefits. Modification of fatty acid content can be used to achievebeneficial levels or profiles of desired PUFA's in plants, plant parts,and plant products, including plant seed oils. For example, when thedesired PUFA's are produced in the seed tissue of a plant, the oil maybe isolated from the seeds typically resulting in an oil high in desiredPUFAs or an oil having a desired fatty acid content or profile, whichmay in turn be used to provide beneficial characteristics in food stuffsand other products. The invention in particular embodiments providesendogenous soybean oil having SDA while also containing a beneficialoleic acid content.

Various aspects of the invention include methods and compositions formodification of PUFA content of a cell, for example, modification of thePUFA content of a plant cell(s). Compositions related to the inventioninclude novel isolated polynucleotide sequences, polynucleotideconstructs and plants and/or plant parts transformed by polynucleotidesof the invention. The isolated polynucleotide may encode Primula fattyacid desaturases and, in particular, may encode a Primula Δ6-desaturase.Host cells may be manipulated to express a polynucleotide encoding adesaturase polypeptide(s) which catalyze desaturation of a fattyacid(s).

Some aspects of the invention include desaturase polypeptides andpolynucleotides encoding the same. Various embodiments of the inventionmay use combinations of desaturase polynucleotides and the encodedpolypeptides that typically depend upon the host cell, the availabilityof substrate(s), and the desired end product(s). “Desaturase” refers toa polypeptide that can desaturate or catalyze formation of a double bondbetween consecutive carbons of one or more fatty acids to produce amono- or poly-unsaturated fatty acid or a precursor thereof. Ofparticular interest are polypeptides that can catalyze the conversion ofoleic acid to LA, LA to ALA, or ALA to SDA, which includes enzymes whichdesaturate at the 12, 15, or 6 positions. The term “polypeptide” refersto any chain of amino acids, regardless of length or post-translationalmodification (e.g., glycosylation or phosphorylation). Considerationsfor choosing a specific polypeptide having desaturase activity include,but are not limited to, the pH optimum of the polypeptide, whether thepolypeptide is a rate limiting enzyme or a component thereof, whetherthe desaturase used is essential for synthesis of a desired PUFA, and/orwhether a co-factor is required by the polypeptide. The expressedpolypeptide preferably has characteristics that are compatible with thebiochemical environment of its location in the host cell. For example,the polypeptide may have to compete for substrate(s).

Analyses of the K_(m) and specific activity of a polypeptide in questionmay be considered in determining the suitability of a given polypeptidefor modifying PUFA(s) production, level, or profile in a given hostcell. The polypeptide used in a particular situation is one whichtypically can function under the conditions present in the intended hostcell, but otherwise may be any desaturase polypeptide having a desiredcharacteristic or being capable of modifying the relative production,level or profile of a desired PUFA(s) or any other desiredcharacteristics as discussed herein. The substrate(s) for the expressedenzyme may be produced by the host cell or may be exogenously supplied.To achieve expression, the polypeptide(s) of the instant invention areencoded by polynucleotides as described below.

The inventors have isolated and produced enzymes from Primula thatexhibit Δ6-desaturase activity. The sequences encoding the Δ6-desaturasemay be expressed in transgenic plants, microorganisms or animals toeffect greater synthesis of SDA. Other polynucleotides which aresubstantially identical to the Δ6-desaturase polynucleotides providedherein, or which encode polypeptides which are substantially identicalto the Δ6-desaturase polypeptides, also can be used. “Substantiallyidentical” refers to an amino acid sequence or nucleic acid sequenceexhibiting in order of increasing preference at least 90% , 95%, 98 or99% identity to the Δ6-desaturase polypeptide sequence in SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:46 orSEQ ID NO:48 or sequences encoding these polypeptides. Polypeptide orpolynucleotide comparisons may be carried out using sequence analysissoftware, for example, the Sequence Analysis software package of the GCGWisconsin Package (Accelrys, San Diego, Calif.), MEGAlign (DNAStar,Inc., 1228 S. Park St., Madison, Wis. 53715), and MacVector (OxfordMolecular Group, 2105 S. Bascom Avenue, Suite 200, Campbell, Calif.95008). Such software matches similar sequences by assigning degrees ofsimilarity or identity.

Encompassed by the present invention are related desaturases, includingvariants of the disclosed Δ6-desaturases naturally occurring within thesame or different species of Primula. Related desaturases can beidentified by their ability to function substantially the same as thedisclosed desaturases; that is, having Δ6-desaturase activity. Relateddesaturases also can be identified by screening sequence databases forsequences homologous to the disclosed desaturases, by hybridization of aprobe based on the disclosed desaturases to a library constructed fromthe source organism, or by RT-PCR using mRNA from the source organismand primers based on the disclosed desaturases. The invention thereforeprovides nucleic acids hybridizing under stringent conditions to adesaturase coding sequences described herein. One of skill in the artunderstands that conditions may be rendered less stringent by increasingsalt concentration and decreasing temperature. Thus, hybridizationconditions can be readily manipulated, and thus will generally be amethod of choice depending on the desired results. An example of highstringency conditions is 5×SSC, 50% formamide and 42° C. By conducting awash under such conditions, for example, for 10 minutes, those sequencesnot hybridizing to a particular target sequence under these conditionscan be removed.

In another aspect of the invention, vectors containing a nucleic acid,or fragment thereof, containing a promoter, a Δ6-desaturase codingsequence and a termination region may be transferred into an organism inwhich the promoter and termination regions are functional. Accordingly,organisms producing recombinant Δ6-desaturase are provided by thisinvention. Yet another aspect of this invention provides isolatedΔ6-desaturase, which can be purified from the recombinant organisms bystandard methods of protein purification. (For example, see Ausubel etal., 1994).

Various aspects of the invention include nucleic acid sequences thatencode desaturases, described herein. Nucleic acids may be isolated fromPrimula including SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:45 or SEQ ID NO:47 and the like. A cloningstrategy based on oligonucleotide primers designed to amplify sequencesidentified as potential fatty acid desaturases, based on BLAST searchesof genomic DNA databases, may be used to sequence individual clones.These clones may then be functionally characterized.

Nucleic acid constructs may be provided that integrate into the genomeof a host cell or are autonomously replicated (e.g., episomallyreplicated) in the host cell. For production of ALA and/or SDA, theexpression cassettes (i.e., a polynucleotide encoding a protein that isoperatively linked to nucleic acid sequence(s) that directs theexpression of the polynucleotide) generally used include an expressioncassette which provides for expression of a polynucleotide encoding aΔ6-desaturase. In certain embodiments a host cell may have wild typeoleic acid content.

Methods and compositions for the construction of expression vectors,when taken in light of the teachings provided herein, for expression ofPrimula desaturase enzymes will be apparent to one of ordinary skill inthe art. Expression vectors, as described herein, are DNA or RNAmolecules engineered for controlled expression of a desiredpolynucleotide, e.g., the Δ6-desaturase-encoding polynucleotide.Examples of vectors include plasmids, bacteriophages, cosmids orviruses. Shuttle vectors, e.g. (Wolk et al. 1984; Bustos et al., 1991)are also contemplated in accordance with the present invention. Reviewsof vectors and methods of preparing and using them can be found inSambrook et al. (2001); Goeddel (1990); and Perbal (1988). Sequenceelements capable of effecting expression of a polynucleotide includepromoters, enhancer elements, upstream activating sequences,transcription termination signals and polyadenylation sites.

Polynucleotides encoding desaturases may be placed under transcriptionalcontrol of a strong promoter. In some cases this leads to an increase inthe amount of desaturase enzyme expressed and concomitantly an increasein the fatty acid produced as a result of the reaction catalyzed by theenzyme. There are a wide variety of plant promoter sequences which maybe used to drive tissue-specific expression of polynucleotides encodingdesaturases in transgenic plants. Indeed, in particular embodiments ofthe invention, the promoter used is a seed specific promoter. Examplesof such promoters include the 5′ regulatory regions from such genes asnapin (Kridl et al., Seed Sci. Res. 1:209:219, 1991), phaseolin (Bustos,et al., Plant Cell, 1(9):839-853, 1989), soybean trypsin inhibitor(Riggs, et al., Plant Cell 1(6):609-621, 1989), ACP (Baerson et al.,Plant Mol. Biol., 22(2):255-267, 1993), stearoyl-ACP desaturase(Slocombe et al., Plant Physiol. 104(4):167-176, 1994), soybean a′subunit of β-conglycinin (P-Gm7S, see for example, Chen et al., Proc.Natl. Acad. Sci. 83:8560-8564, 1986), Vicia faba USP (P-Vf.Usp, see forexample, SEQ ID NO:1, 2, and 3, U.S. patent application Ser. No.10/429,516), the globulin promoter (see for example Belanger and Kriz,Genet. 129: 863-872 (1991), soybean alpha subunit of β-conglycinin (7Salpha) (U.S. patent application Ser. No. 10/235,618, incorporated byreference) and Zea mays L3 oleosin promoter (P-Zm.L3, see, for example,Hong et al., Plant Mol. Biol., 34(3):549-555, 1997). Also included arethe zeins, which are a group of storage proteins found in cornendosperm. Genomic clones for zein genes have been isolated (Pedersen etal., Cell 29:1015-1026 (1982), and Russell et al., Transgenic Res.6(2):157-168) and the promoters from these clones, including the 15 kD,16 kD, 19 kD, 22 kD, 27 kD and genes, could also be used.

The ordinarily skilled artisan can determine vectors and regulatoryelements (including operably linked promoters and coding regions)suitable for expression in a particular host cell. “Operably linked” inthis context means that the promoter and terminator sequenceseffectively function to regulate transcription. As a further example, avector appropriate for expression of Δ6-desaturase in transgenic plantscan comprise a seed-specific promoter sequence derived fromhelianthinin, napin, or glycinin operably linked to the Δ6-desaturasecoding region and further operably linked to a seed storage proteintermination signal or the nopaline synthase termination signal. As astill further example, a vector for use in expression of Δ6-desaturasein plants can comprise a constitutive promoter or a tissue specificpromoter operably linked to the Δ6-desaturase coding region and furtheroperably linked to a constitutive or tissue specific terminator or thenopaline synthase termination signal.

Modifications of the nucleotide sequences or regulatory elementsdisclosed herein which maintain the functions contemplated herein arewithin the scope of this invention. Such modifications includeinsertions, substitutions and deletions, and specifically substitutionswhich reflect the degeneracy of the genetic code.

Standard techniques for the construction of such recombinant vectors arewell-known to those of ordinary skill in the art and can be found inreferences such as Sambrook et al. (2001), or any of the myriad oflaboratory manuals on recombinant DNA technology that are widelyavailable. A variety of strategies are available for ligating fragmentsof DNA, the choice of which depends on the nature of the termini of theDNA fragments. It is further contemplated in accordance with the presentinvention to include in a nucleic acid vector other nucleotide sequenceelements which facilitate cloning, expression or processing, for examplesequences encoding signal peptides, a sequence encoding KDEL, which isrequired for retention of proteins in the endoplasmic reticulum orsequences encoding transit peptides which direct Δ6-desaturase to thechloroplast. Such sequences are known to one of ordinary skill in theart. An optimized transit peptide is described, for example, by Van denBroeck et al. (1985). Prokaryotic and eukaryotic signal sequences aredisclosed, for example, by Michaelis et al. (1982).

Polynucleotides encoding desired desaturases can be identified in avariety of ways. As an example, a source of the desired desaturase, forexample genomic or cDNA libraries from Primula, is screened withdetectable enzymatically- or chemically-synthesized probes, which can bemade from DNA, RNA, or non-naturally occurring nucleotides, or mixturesthereof. Probes may be enzymatically synthesized from polynucleotides ofknown desaturases for normal or reduced-stringency hybridizationmethods. Oligonucleotide probes also can be used to screen sources andcan be based on sequences of known desaturases, including sequencesconserved among known desaturases, or on peptide sequences obtained fromthe desired purified protein. Oligonucleotide probes based on amino acidsequences can be degenerate to encompass the degeneracy of the geneticcode, or can be biased in favor of the preferred codons of the sourceorganism. Oligonucleotides also can be used as primers for PCR fromreverse transcribed mRNA from a known or suspected source; the PCRproduct can be the full length cDNA or can be used to generate a probeto obtain the desired full length cDNA. Alternatively, a desired proteincan be entirely sequenced and total synthesis of a DNA encoding thatpolypeptide performed.

Once the desired genomic or cDNA has been isolated, it can be sequencedby known methods. It is recognized in the art that such methods aresubject to errors, such that multiple sequencing of the same region isroutine and is still expected to lead to measurable rates of mistakes inthe resulting deduced sequence, particularly in regions having repeateddomains, extensive secondary structure, or unusual base compositions,such as regions with high GC base content. When discrepancies arise,resequencing can be done and can employ special methods. Special methodscan include altering sequencing conditions by using: differenttemperatures; different enzymes; proteins which alter the ability ofoligonucleotides to form higher order structures; altered nucleotidessuch as ITP or methylated dGTP; different gel compositions, for exampleadding formamide; different primers or primers located at differentdistances from the problem region; or different templates such as singlestranded DNAs. Sequencing of mRNA also can be employed.

Some or all of the coding sequence for a polypeptide having desaturaseactivity may be from a natural source. In some situations, however, itis desirable to modify all or a portion of the codons, for example, toenhance expression, by employing host preferred codons. Host-preferredcodons can be determined from the codons of highest frequency in theproteins expressed in the largest amount in a particular host speciesand/or tissue of interest. Thus, the coding sequence for a polypeptidehaving desaturase activity can be synthesized in whole or in part. Allor portions of the DNA also can be synthesized to remove anydestabilizing sequences or regions of secondary structure which would bepresent in the transcribed mRNA. All or portions of the DNA also can besynthesized to alter the base composition to one more preferable in thedesired host cell. Methods for synthesizing sequences and bringingsequences together are well established in the literature. In vitromutagenesis and selection, site-directed mutagenesis, or other means canbe employed to obtain mutations of naturally-occurring desaturase genesto produce a polypeptide having desaturase activity in vivo with moredesirable physical and kinetic parameters for function in the host cell,such as a longer half-life or a higher rate of production of a desiredpolyunsaturated fatty acid.

Once the polynucleotide encoding a desaturase polypeptide has beenobtained, it is placed in a vector capable of replication in a hostcell, or is propagated in vitro by means of techniques such as PCR orlong PCR. Replicating vectors can include plasmids, phage, viruses,cosmids and the like. Desirable vectors include those useful formutagenesis of the gene of interest or for expression of the gene ofinterest in host cells. The technique of long PCR has made in vitropropagation of large constructs possible, so that modifications to thegene of interest, such as mutagenesis or addition of expression signals,and propagation of the resulting constructs can occur entirely in vitrowithout the use of a replicating vector or a host cell.

For expression of a desaturase polypeptide, functional transcriptionaland translational initiation and termination regions are operably linkedto the polynucleotide encoding the desaturase polypeptide. Expression ofthe polypeptide coding region can take place in vitro or in a host cell.Transcriptional and translational initiation and termination regions arederived from a variety of nonexclusive sources, including thepolynucleotide to be expressed, genes known or suspected to be capableof expression in the desired system, expression vectors, chemicalsynthesis, or from an endogenous locus in a host cell.

Expression in a host cell can be accomplished in a transient or stablefashion. Transient expression can occur from introduced constructs whichcontain expression signals functional in the host cell, but whichconstructs do not replicate and rarely integrate in the host cell, orwhere the host cell is not proliferating. Transient expression also canbe accomplished by inducing the activity of a regulatable promoteroperably linked to the gene of interest, although such inducible systemsfrequently exhibit a low basal level of expression. Stable expressioncan be achieved by introduction of a construct that can integrate intothe host genome or that autonomously replicates in the host cell. Stableexpression of the gene of interest can be selected for through the useof a selectable marker located on or transfected with the expressionconstruct, followed by selection for cells expressing the marker. Whenstable expression results from integration, integration of constructscan occur randomly within the host genome or can be targeted through theuse of constructs containing regions of homology with the host genomesufficient to target recombination with the host locus. Where constructsare targeted to an endogenous locus, all or some of the transcriptionaland translational regulatory regions can be provided by the endogenouslocus.

When increased expression of the desaturase polypeptide in the sourceorganism is desired, several methods can be employed. Additional genesencoding the desaturase polypeptide can be introduced into the hostorganism. Expression from the native desaturase locus also can beincreased through homologous recombination, for example by inserting astronger promoter into the host genome to cause increased expression, byremoving destabilizing sequences from either the mRNA or the encodedprotein by deleting that information from the host genome, or by addingstabilizing sequences to the mRNA (U.S. Pat. No. 4,910,141).

It is contemplated that more than one polynucleotide encoding adesaturase or a polynucleotide encoding more than one desaturase may beintroduced and propagated in a host cell through the use of episomal orintegrated expression vectors. Where two or more genes are expressedfrom separate replicating vectors, it is desirable that each vector hasa different means of replication. Each introduced construct, whetherintegrated or not, should have a different means of selection and shouldlack homology to the other constructs to maintain stable expression andprevent reassortment of elements among constructs. Judicious choices ofregulatory regions, selection means and method of propagation of theintroduced construct can be experimentally determined so that allintroduced polynucleotides are expressed at the necessary levels toprovide for synthesis of the desired products.

When necessary for transformation, the Δ6-desaturase coding sequences ofthe present invention can be inserted into a plant transformationvector, e.g. the binary vector described by Bevan (1984). Planttransformation vectors can be derived by modifying the natural genetransfer system of Agrobacterium tumefaciens. The natural systemcomprises large Ti (tumor-inducing)-plasmids containing a large segment,known as T-DNA, which is transferred to transformed plants. Anothersegment of the Ti plasmid, the vir region, is responsible for T-DNAtransfer. The T-DNA region is bordered by terminal repeats. In themodified binary vectors the tumor-inducing genes have been deleted andthe functions of the vir region are utilized to transfer foreign DNAbordered by the T-DNA border sequences. The T-region also contains aselectable marker for antibiotic resistance, and a multiple cloning sitefor inserting sequences for transfer. Such engineered strains are knownas “disarmed” A. tumefaciens strains, and allow the efficienttransformation of sequences bordered by the T-region into the nucleargenomes of plants.

The subject invention finds many applications. Probes based on thepolynucleotides of the present invention may find use in methods forisolating related molecules or in methods to detect organisms expressingdesaturases. When used as probes, the polynucleotides oroligonucleotides must be detectable. This is usually accomplished byattaching a label either at an internal site, for example viaincorporation of a modified residue, or at the 5′ or 3′ terminus. Suchlabels can be directly detectable, can bind to a secondary molecule thatis detectably labeled, or can bind to an unlabelled secondary moleculeand a detectably labeled tertiary molecule; this process can be extendedas long as is practical to achieve a satisfactorily detectable signalwithout unacceptable levels of background signal. Secondary, tertiary,or bridging systems can include use of antibodies directed against anyother molecule, including labels or other antibodies, or can involve anymolecules which bind to each other, for example abiotin-streptavidin/avidin system. Detectable labels typically includeradioactive isotopes, molecules which chemically or enzymaticallyproduce or alter light, enzymes which produce detectable reactionproducts, magnetic molecules, fluorescent molecules or molecules whosefluorescence or light-emitting characteristics change upon binding.Examples of labeling methods can be found in U.S. Pat. No. 5,011,770.Alternatively, the binding of target molecules can be directly detectedby measuring the change in heat of solution on binding of probe totarget via isothermal titration calorimetry, or by coating the probe ortarget on a surface and detecting the change in scattering of light fromthe surface produced by binding of target or probe, respectively, as maybe done with the BIAcore system.

Constructs comprising the gene of interest may be introduced into a hostcell by standard techniques. For convenience, a host cell which has beenmanipulated by any method to take up a DNA sequence or construct will bereferred to as “transformed” or “recombinant” herein. The subject hostwill have at least have one copy of the expression construct and mayhave two or more, for example, depending upon whether the gene isintegrated into the genome, amplified, or is present on anextrachromosomal element having multiple copy numbers.

The transformed host cell can be identified by selection for a markercontained on the introduced construct. Alternatively, a separate markerconstruct may be introduced with the desired construct, as manytransformation techniques introduce many DNA molecules into host cells.Typically, transformed hosts are selected for their ability to grow onselective media. Selective media may incorporate an antibiotic or lack afactor necessary for growth of the untransformed host, such as anutrient or growth factor. An introduced marker gene therefore mayconfer antibiotic resistance, or encode an essential growth factor orenzyme, and permit growth on selective media when expressed in thetransformed host. Selection of a transformed host can also occur whenthe expressed marker protein can be detected, either directly orindirectly. The marker protein may be expressed alone or as a fusion toanother protein. The marker protein can be detected by its enzymaticactivity; for example, beta-galactosidase can convert the substrateX-gal to a colored product, and luciferase can convert luciferin to alight-emitting product. The marker protein can be detected by itslight-producing or modifying characteristics; for example, the greenfluorescent protein of Aequorea victoria fluoresces when illuminatedwith blue light. Antibodies can be used to detect the marker protein ora molecular tag on, for example, a protein of interest. Cells expressingthe marker protein or tag can be selected, for example, visually, or bytechniques such as FACS or panning using antibodies. Desirably,resistance to kanamycin and the amino glycoside G418 are of interest, aswell as ability to grow on media lacking uracil, leucine, lysine ortryptophan.

Of particular interest is the Δ6-desaturase-mediated production ofPUFA's in eukaryotic host cells. Eukaryotic cells include plant cells,such as those from oil-producing crop plants, and other cells amenableto genetic manipulation including fungal cells. The cells may becultured or formed as part or all of a host organism including a plant.In a preferred embodiment, the host is a plant cell which producesand/or can assimilate exogenously supplied substrate(s) for aΔ6-desaturase, and preferably produces large amounts of one or more ofthe substrates.

The transformed host cell is grown under appropriate conditions adaptedfor a desired end result. For host cells grown in culture, theconditions are typically optimized to produce the greatest or mosteconomical yield of PUFA's, which relates to the selected desaturaseactivity. Media conditions which may be optimized include: carbonsource, nitrogen source, addition of substrate, final concentration ofadded substrate, form of substrate added, aerobic or anaerobic growth,growth temperature, inducing agent, induction temperature, growth phaseat induction, growth phase at harvest, pH, density, and maintenance ofselection.

Another aspect of the present invention provides transgenic plants orprogeny of plants containing the isolated DNA of the invention. Bothmonocotyledonous and dicotyledonous plants are contemplated. Plant cellsare transformed with an isolated DNA encoding Δ6-desaturase by any planttransformation method. The transformed plant cell, often in a callusculture or leaf disk, is regenerated into a complete transgenic plant bymethods well-known to one of ordinary skill in the art (e.g. Horsch etal., 1985). In one embodiment, the transgenic plant is selected from thegroup consisting of Arabidopsis thaliana, canola, soy, soybean,rapeseed, sunflower, cotton, cocoa, peanut, safflower, coconut, flax,oil palm, oilseed Brassica napus, corn, jojoba, Chinese tallow tree,tobacco, fruit plants, citrus plants or plants producing nuts andberries. Since progeny of transformed plants inherit the polynucleotideencoding Δ6-desaturase, seeds or cuttings from transformed plants may beused to maintain the transgenic plant line.

The present invention further provides a method for providing transgenicplants with an increased content of ALA and/or SDA. This methodincludes, for example, introducing DNA encoding Δ6-desaturase into plantcells which lack or have low levels SDA but contain ALA, andregenerating plants with increased SDA content from the transgeniccells. In certain embodiments of the invention, a DNA encoding a Δ15-and/or Δ12-desaturase may also be introduced into the plant cells. Suchplants may or may not also comprise endogenous Δ12- and/orΔ15-desaturase activity. In certain embodiments, modified commerciallygrown crop plants are contemplated as the transgenic organism,including, but not limited to, Arabidopsis thaliana, canola, soy,soybean, rapeseed, sunflower, cotton, cocoa, peanut, safflower, coconut,flax, oil palm, oilseed Brassica napus, corn, jojoba, Chinese tallowtree, tobacco, fruit plants, citrus plants or plants producing nuts andberries.

The present invention further provides a method for providing transgenicplants which may contain elevated levels of ALA and/or SDA, wherein saidelevated levels are greater than levels found in non-transformed plants.Expression vectors comprising DNA encoding a Δ6-desaturase, and/or aΔ12-desaturase and/or a Δ15-desaturase, can be constructed by methods ofrecombinant technology known to one of ordinary skill in the art(Sambrook et al., 2001). In particular, commercially grown crop plantsare contemplated as the transgenic organism, including, but not limitedto, Arabidopsis thaliana, canola, soy, soybean, rapeseed, sunflower,cotton, cocoa, peanut, safflower, coconut, flax, oil palm, oilseedBrassica napus, and corn.

For dietary supplementation, the purified PUFAs, transformed plants orplant parts, or derivatives thereof, may be incorporated into cookingoils, fats or margarines formulated so that in normal use the recipientwould receive the desired amount. The PUFAs may also be incorporatedinto infant formulas, nutritional supplements or other food products,and may find use as anti-inflammatory or cholesterol lowering agents.

As used herein, “edible composition” is defined as compositions whichmay be ingested by a mammal such as foodstuffs, nutritional substancesand pharmaceutical compositions. As used herein “foodstuffs” refer tosubstances that can be used or prepared for use as food for a mammal andinclude substances that may be used in the preparation of food (such asfrying oils) or food additives. For example, foodstuffs include animalsused for human consumption or any product therefrom, such as, forexample, eggs. Typical foodstuffs include but are not limited tobeverages, (e.g., soft drinks, carbonated beverages, ready to mixbeverages), infused foods (e.g. fruits and vegetables), sauces,condiments, salad dressings, fruit juices, syrups, desserts (e.g.,puddings, gelatin, icings and fillings, baked goods and frozen dessertssuch as ice creams and sherbets), soft frozen products (e.g., softfrozen creams, soft frozen ice creams and yoghurts, soft frozen toppingssuch as dairy or non-dairy whipped toppings), oils and emulsifiedproducts (e.g., shortening, margarine, mayonnaise, butter, cooking oil,and salad dressings) and intermediate moisture foods (e.g., rice and dogfoods).

Furthermore, edible compositions described herein can also be ingestedas an additive or supplement contained in foods and drinks. These can beformulated together with a nutritional substance such as variousvitamins and minerals and incorporated into substantially liquidcompositions such as nutrient drinks, soymilks and soups; substantiallysolid compositions; and gelatins or used in the form of a powder to beincorporated into various foods. The content of the effective ingredientin such a functional or health food can be similar to the dose containedin a typical pharmaceutical agent.

The purified PUFAs, transformed plants or plant parts may also beincorporated into animal, particularly livestock, feed. In this way, theanimals themselves may benefit from a PUFA rich diet, while humanconsumers of food products produced from such livestock may benefit aswell. It is expected in certain embodiments that SDA will be convertedto EPA in animals and thus such animals may benefit from an increase inEPA by consumption of SDA.

For pharmaceutical use (human or veterinary), the compositions maygenerally be administered orally but can be administered by any route bywhich they may be successfully absorbed, e.g., parenterally (i.e.subcutaneously, intramuscularly or intravenously), rectally, vaginallyor topically, for example, as a skin ointment or lotion. The PUFAstransformed plants or plant parts of the present invention may beadministered alone or in combination with a pharmaceutically acceptablecarrier or excipient. Where available, gelatin capsules are thepreferred form of oral administration. Dietary supplementation as setforth above can also provide an oral route of administration. Theunsaturated acids of the present invention may be administered inconjugated forms, or as salts, esters, amides or prodrugs of the fattyacids. Any pharmaceutically acceptable salt is encompassed by thepresent invention; especially preferred are the sodium, potassium orlithium salts. Also encompassed are the N-alkylpolyhydroxamine salts,such as N-methyl glucamine, found in PCT publication WO 96/33155. Thepreferred esters are the ethyl esters. As solid salts, the PUFAs alsocan be administered in tablet form. For intravenous administration, thePUFAs or derivatives thereof may be incorporated into commercialformulations such as Intralipids.

If desired, the regions of a desaturase polypeptide important fordesaturase activity can be determined through routine mutagenesisfollowed by expression of the resulting mutant polypeptides anddetermination of their activities. Mutants may include substitutions,deletions, insertions and point mutations, or combinations thereof.Substitutions may be made on the basis of conserved hydrophobicity orhydrophilicity (Kyte and Doolittle, 1982), or on the basis of theability to assume similar polypeptide secondary structure (Chou andFasman, 1978). A typical functional analysis begins with deletionmutagenesis to determine the N- and C-terminal limits of the proteinnecessary for function, and then internal deletions, insertions or pointmutants are made to further determine regions necessary for function.Other techniques such as cassette mutagenesis or total synthesis alsocan be used. Deletion mutagenesis is accomplished, for example, by usingexonucleases to sequentially remove the 5′ or 3′ coding regions. Kitsare available for such techniques. After deletion, the coding region iscompleted by ligating oligonucleotides containing start or stop codonsto the deleted coding region after 5′ or 3′ deletion, respectively.Alternatively, oligonucleotides encoding start or stop codons areinserted into the coding region by a variety of methods includingsite-directed mutagenesis, mutagenic PCR or by ligation onto DNAdigested at existing restriction sites.

Internal deletions can similarly be made through a variety of methodsincluding the use of existing restriction sites in the DNA, by use ofmutagenic primers via site directed mutagenesis or mutagenic PCR.Insertions are made through methods such as linker-scanning mutagenesis,site-directed mutagenesis or mutagenic PCR. Point mutations are madethrough techniques such as site-directed mutagenesis or mutagenic PCR.Chemical mutagenesis may also be used for identifying regions of adesaturase polypeptide important for activity. Such structure-functionanalysis can determine which regions may be deleted, which regionstolerate insertions, and which point mutations allow the mutant proteinto function in substantially the same way as the native desaturase. Allsuch mutant proteins and nucleotide sequences encoding them are withinthe scope of the present invention.

As described herein above, certain embodiments of the current inventionconcern plant transformation constructs. For example, one aspect of thecurrent invention is a plant transformation vector comprising one ormore desaturase gene(s) or cDNA(s). Exemplary coding sequences for usewith the invention include Primula juliae Δ6-desaturase (SEQ IDNOs:2-3). In certain embodiments, antisense desaturase sequences canalso be employed with the invention. Exemplary desaturase encodingnucleic acids include at least 20, 40, 80, 120, 300 and up to the fulllength of the nucleic acid sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:45 or SEQ ID NO:47. Incertain aspects, a nucleic acid may encode 1, 2, 3, 4, or moredesaturase enzymes. In particular embodiments, a nucleic acid may encodea Δ6- and a Δ15-desaturase.

Vectors used for plant transformation may include, for example,plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterialartificial chromosomes) or any other suitable cloning system, as well asfragments of DNA therefrom. Thus when the term “vector” or “expressionvector” is used, all of the foregoing types of vectors, as well asnucleic acid sequences isolated therefrom, are included. It iscontemplated that utilization of cloning systems with large insertcapacities will allow introduction of large DNA sequences comprisingmore than one selected gene. In accordance with the invention, thiscould be used to introduce various desaturase encoding nucleic acids.Introduction of such sequences may be facilitated by use of bacterial oryeast artificial chromosomes (BACs or YACs, respectively), or even plantartificial chromosomes. For example, the use of BACs forAgrobacterium-mediated transformation was disclosed by Hamilton et al.(1996).

Particularly useful for transformation are expression cassettes whichhave been isolated from such vectors. DNA segments used for transformingplant cells will, of course, generally comprise the cDNA, gene or geneswhich one desires to introduce into and have expressed in the hostcells. These DNA segments can further include structures such aspromoters, enhancers, polylinkers, or even regulatory genes as desired.The DNA segment or gene chosen for cellular introduction will oftenencode a protein which will be expressed in the resultant recombinantcells resulting in a screenable or selectable trait and/or which willimpart an improved phenotype to the resulting transgenic plant. However,this may not always be the case, and the present invention alsoencompasses transgenic plants incorporating non-expressed transgenes.Preferred components likely to be included with vectors used in thecurrent invention are as follows.

In one embodiment the instant invention utilizes certain promoters.Examples of such promoters that may be used with the instant inventioninclude, but are not limited to, the 35S CaMV (cauliflower mosaicvirus), 34S FMV (figwort mosaic virus) (see, e.g., U.S. Pat. No.5,378,619, the contents of which are herein incorporated in theirentirety), Napin (from Brassica), 7S (from soybean), Globulin and Lec(from corn). The napin promoter and promoters, which are regulatedduring plant seed maturation, are of particular interest for use withthe instant invention. All such promoter and transcriptional regulatoryelements, singly or in combination, are contemplated for use in thepresent replicable expression vectors and are known to one of ordinaryskill in the art.

The DNA sequence between the transcription initiation site and the startof the coding sequence, i.e., the untranslated leader sequence, can alsoinfluence gene expression. One may thus wish to employ a particularleader sequence with a transformation construct of the invention.Preferred leader sequences are contemplated to include those whichcomprise sequences predicted to direct optimum expression of theattached gene, i.e., to include a preferred consensus leader sequencewhich may increase or maintain mRNA stability and prevent inappropriateinitiation of translation. The choice of such sequences will be known tothose of skill in the art in light of the present disclosure. Sequencesthat are derived from genes that are highly expressed in plants willtypically be preferred.

Transformation constructs prepared in accordance with the invention willtypically include a 3′ end DNA sequence that acts as a signal toterminate transcription and allow for the poly-adenylation of the mRNAproduced by coding sequences operably linked to a desaturase gene (e.g.,cDNA). In one embodiment of the invention, the native terminator of adesaturase gene is used. Alternatively, a heterologous 3′ end mayenhance the expression of desaturase coding regions. Examples ofterminators deemed to be useful include those from the nopaline synthasegene of Agrobacterium tumefaciens (nos 3′ end) (Bevan et al., 1983), theterminator for the T7 transcript from the octopine synthase gene ofAgrobacterium tumefaciens, the 3′ end of the protease inhibitor I or IIgenes from potato or tomato and the CaMV 35S terminator (tml3′).Regulatory elements such as an Adh intron (Callis et al., 1987), sucrosesynthase intron (Vasil et al., 1989) or TMV omega element (Gallie etal., 1989), may further be included where desired.

By employing a selectable or screenable marker protein, one can provideor enhance the ability to identify transformants. “Marker genes” aregenes that impart a distinct phenotype to cells expressing the markerprotein and thus allow such transformed cells to be distinguished fromcells that do not have the marker. Such genes may encode either aselectable or screenable marker, depending on whether the marker confersa trait which one can “select” for by chemical means, i.e., through theuse of a selective agent (e.g., a herbicide, antibiotic, or the like),or whether it is simply a trait that one can identify throughobservation or testing, i.e., by “screening” (e.g., the greenfluorescent protein). Of course, many examples of suitable markerproteins are known to the art and can be employed in the practice of theinvention.

Suitable methods for transformation of plant or other cells for use withthe current invention are believed to include virtually any method bywhich DNA can be introduced into a cell, such as by direct delivery ofDNA such as by PEG-mediated transformation of protoplasts (Omirulleh etal., 1993), by desiccation/inhibition-mediated DNA uptake (Potrykus etal., 1985), by electroporation (U.S. Pat. No. 5,384,253, specificallyincorporated herein by reference in its entirety), by agitation withsilicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. No. 5,302,523,specifically incorporated herein by reference in its entirety; and U.S.Pat. No. 5,464,765, specifically incorporated herein by reference in itsentirety), by Agrobacterium-mediated transformation (U.S. Pat. No.5,591,616 and U.S. Pat. No. 5,563,055; both specifically incorporatedherein by reference) and by acceleration of DNA coated particles (U.S.Pat. No. 5,550,318; U.S. Pat. No. 5,538,877; and U.S. Pat. No.5,538,880; each specifically incorporated herein by reference in itsentirety), etc. Through the application of techniques such as these, thecells of virtually any plant species may be stably transformed, andthese cells developed into transgenic plants.

After effecting delivery of exogenous DNA to recipient cells, the nextsteps generally concern identifying the transformed cells for furtherculturing and plant regeneration. In order to improve the ability toidentify transformants, one may desire to employ a selectable orscreenable marker gene with a transformation vector prepared inaccordance with the invention. In this case, one would then generallyassay the potentially transformed cell population by exposing the cellsto a selective agent or agents, or one would screen the cells for thedesired marker gene trait.

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, may be cultured in mediathat supports regeneration of plants. In an exemplary embodiment, MS andN6 media may be modified by including further substances such as growthregulators. One such growth regulator is dicamba or 2,4-D. However,other growth regulators may be employed, including NAA, NAA+2,4-D orpicloram. Media improvement in these and like ways has been found tofacilitate the growth of cells at specific developmental stages. Tissuemay be maintained on a basic media with growth regulators untilsufficient tissue is available to begin plant regeneration efforts, orfollowing repeated rounds of manual selection, until the morphology ofthe tissue is suitable for regeneration, typically at least 2 weeks,then transferred to media conducive to maturation of embryoids. Culturesare transferred every 2 weeks on this medium. Shoot development willsignal the time to transfer to medium lacking growth regulators.

To confirm the presence of the exogenous DNA or “transgene(s)” in theregenerating plants, a variety of assays may be performed. Such assaysinclude, for example, “molecular biological” assays, such as Southernand Northern blotting and PCR™; “biochemical” assays, such as detectingthe presence of a protein product, e.g., by immunological means (ELISAsand Western blots) or by enzymatic function; plant part assays, such asleaf or root assays; and also, by analyzing the phenotype of the wholeregenerated plant.

In addition to direct transformation of a particular plant genotype witha construct prepared according to the current invention, transgenicplants may be made by crossing a plant having a selected DNA of theinvention to a second plant lacking the DNA. Plant breeding techniquesmay also be used to introduce a multiple desaturases, for example Δ6,Δ12, and/or Δ15-desaturase(s) into a single plant. In this manner,Δ6-desaturase can be effectively up-regulated. By creating plantshomozygous for a Δ6-desaturase activity and/or other desaturase activity(e.g., Δ12- and/or Δ15-desaturase activity) beneficial metabolites canbe increased in the plant.

As set forth above, a selected desaturase gene can be introduced into aparticular plant variety by crossing, without the need for ever directlytransforming a plant of that given variety. Therefore, the currentinvention not only encompasses a plant directly transformed orregenerated from cells which have been transformed in accordance withthe current invention, but also the progeny of such plants. As usedherein the term “progeny” denotes the offspring of any generation of aparent plant prepared in accordance with the instant invention, whereinthe progeny comprises a selected DNA construct prepared in accordancewith the invention. “Crossing” a plant to provide a plant line havingone or more added transgenes or alleles relative to a starting plantline, as disclosed herein, is defined as the techniques that result in aparticular sequence being introduced into a plant line by crossing astarting line with a donor plant line that comprises a transgene orallele of the invention. To achieve this one could, for example, performthe following steps: (a) plant seeds of the first (starting line) andsecond (donor plant line that comprises a desired transgene or allele)parent plants; (b) grow the seeds of the first and second parent plantsinto plants that bear flowers; (c) pollinate a flower from the firstparent plant with pollen from the second parent plant; and (d) harvestseeds produced on the parent plant bearing the fertilized flower.

Backcrossing is herein defined as the process including the steps of:(a) crossing a plant of a first genotype containing a desired gene, DNAsequence or element to a plant of a second genotype lacking said desiredgene, DNA sequence or element; (b) selecting one or more progeny plantcontaining the desired gene, DNA sequence or element; (c) crossing theprogeny plant to a plant of the second genotype; and (d) repeating steps(b) and (c) for the purpose of transferring a desired DNA sequence froma plant of a first genotype to a plant of a second genotype.

Introgression of a DNA element into a plant genotype is defined as theresult of the process of backcross conversion. A plant genotype intowhich a DNA sequence has been introgressed may be referred to as abackcross converted genotype, line, inbred, or hybrid. Similarly a plantgenotype lacking the desired DNA sequence may be referred to as anunconverted genotype, line, inbred, or hybrid.

EXAMPLES

The following examples are included to illustrate embodiments of theinvention. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the concept, spirit and scope ofthe invention. More specifically, it will be apparent that certainagents which are both chemically and physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1

Cloning of Primula juliae Δ6 Desaturase Sequences

Cloning of the Primula juliae Δ6 desaturase (PjD6D) was achieved by PCRamplification of a partial internal genomic DNA region using degenerateoligonucleotides, followed by bi-directional genomic walking. Totalgenomic DNA was isolated from P. juliae (Collector's Nursery,Battleground Wash.) using the DNeasy Plant Mini Kit (Qiagen, Valencia,Calif.), following the manufacturer's procedure. Initially, a 552 bpfragment corresponding to positions 687 to 1238 of SEQ ID NO:1 wasisolated using degenerate oligonucleotides BO-1 For and BO-2 Rev asdescribed by Garcia-Maroto et al. (2002). The fragment was cloned intopCR®4-TOPO® (Invitrogen, Carlsbad, Calif.) to yield the vector pMON83955and the insert was sequenced. Primer sequences BO-1 For and BO-2 Revwere as follows:

BO-1 For: 5′-ATMAGYATYGGTTGGTGGAARTGG-3′ (SEQ ID NO: 6) BO-2 Rev:5′-AATCCACCRTGRAACCARTCCAT-3′ (SEQ ID NO: 7)

To determine the genomic flanking sequence of the insert of pMON83955, aUniversal Genome Walker Kit™ (BD Biosciences, Palo Alto, Calif.) wasutilized, following the manufacture's procedure. Four P. juliae genomiclibraries were generated by digesting the DNA with four restrictionenzymes: EcoRV, PvuII, StuI, and DraI. After a purification step, thedigestions were ligated to an adapter provided in the kit. The procedurethen involved two PCR reactions, each with a gene-specific primer and anadapter-primer. The secondary PCR reaction used a dilution of theprimary PCR reaction products as a template. For the 5′ direction,primers PD6D R8 and PD6D R2 were used for the primary and secondary PCRreactions, respectively. For the 3′ direction, primers PD6D F8 and PD6DF3 were used for the primary and secondary PCR reactions, respectively.The primer sequences are given below:

PD6D R8: 5′-CACACATGACCGGATAAAACGACCAGT-3′ (SEQ ID NO: 8) PD6D R2:5′-GGGAATGTACTGGAGGTCAGGGTCGTA-3′ (SEQ ID NO: 9) PD6D F8:5′-CGTGCAGTTCAGCTTGAACCATTTCTC-3′ (SEQ ID NO: 10) PD6D F3:5′-TGCAGGGACACTCAACATATCGTGCCC-3′ (SEQ ID NO: 11)

Genome walking in the 5′ direction yielded a 574 bp fragment from theEcoRV library. This product was cloned into pCR®4-TOPO® (Invitrogen)giving pMON83956, and the insert was sequenced. The resulting sequencedid not contain a start codon of the putative delta 6 desaturase geneand thus another set of PCR reactions was performed using gene specificprimers designed to walk in the 5′ direction from the pMON83956 insert.The primers used for the second genome walking set in the 5′ directionwere PD6D R15 and PD6D R14 for the primary and secondary PCR reactions,respectively. The sequences are given below:

PD6D R15: 5′-GTAGGTTGGTGGAGAAGGGAGGGAGGA-3′ (SEQ ID NO: 12) PD6D R14:5′-GGAAGGGGGATGGTAAGCGAGGAAAGC-3′ (SEQ ID NO: 13)

A product of 328 bp in length from the StuI library was cloned intopCR®4-TOPO® (Invitrogen) giving pMON83958 and the insert was sequenced.This insert contained 2 potential start codons, 44 bases apart. Thefirst start codon corresponds to position 87 and the second to position135 of SEQ ID NO:1. Genome walking in the 3′ direction resulted in a 773bp fragment from the DraI library. This product was cloned intopCR®4-TOPO®, giving pMON83957. The insert was sequenced and found tocontain 292 bp of the coding region for the putative delta 6 desaturasegene, followed by a stop codon at position 1473 with respect to SEQ IDNO:1.

The inserts of pMON83955, pMON83956, pMON83957, and pMON83958 werealigned to form a composite sequence, SEQ ID NO:1. Three primers weredesigned to PCR amplify 2 different lengths of coding sequence from P.juliae genomic DNA, reflecting the two start codons found in pMON83958.The longer of the two sequences, PjD6D-1, was amplified using forwardprimer Pj D6D F2 and reverse primer Pj D6D R1. The shorter of the two,PjD6D-2, was amplified using forward primer Pj D6D F1 and reverse primerPj D6D R1. The two putative delta 6 desaturase coding sequences wereeach then ligated into the yeast expression vector pYES2.1-TOPO. Uponsequencing, the plasmid containing PjD6D-1 was designated pMON83950 (SEQID NO:3) and the plasmid containing PjD6D-2 was designated pMON67011(SEQ ID NO:2). The primer sequences are given below:

Pj D6D F2: 5′-GTCGACATGGAAAACACATTTTCACCACCACCT-3′ (SEQ ID NO: 14) PjD6D F1: 5′-GTCGACATGACTAAGACGATTTACATAACCAGC-3′ (SEQ ID NO: 15) Pj D6DR1: 5′-CCTGCAGGTCACCCGACATTTTTAACAGCCTCCC-3′ (SEQ ID NO: 16)

The two PjΔ6 desaturase clones, PjD6D-2 and PjD6D-1, encode potentialpolypeptides of 446 amino acids and 462 amino acids, given in SEQ IDNO:4 and SEQ ID NO:5, respectively. The initial MET site of the shorterpeptide sequence (PjD6D-2) is located 16 amino acids downstream from thefirst MET site of the longer sequence (PjD6D-1). 3′ of the second MET,the sequences are identical. These sequences have high similarity toother plant Δ6 desaturases (FIG. 1), including an N-terminal cytochromeb₅ domain which is found in all front-end desaturases (Napier et al.,2003). Within the cytochrome b₅ domain is found the eight invariantresidues characteristic of the cytochrome b₅ superfamily and the H-P-G-Gheme-binding motif, which has been shown to be essential for enzymaticactivity (Napier et al., 1997, Sayanova et al, 1999, Sperling and Heinz2001). Within the desaturase domain of the putative PjD6D desaturase arethree conserved histidine boxes that are characteristic of allmembrane-bound desaturases (Shanklin et al., 1994). A distinguishingfeature found in all front-end desaturases is that the third histidinebox contains a glutamine residue in the first position (Q-x-x-H-H)instead of a histidine (Napier et al., 1997, Napier et al., 2003,Sperling and Heinz 2001). The deduced amino acid sequence of the PjD6Dhad approximately 88% identity to the Primula vialii and P. farinosadesaturases and approximately 64% identity to the Echium pitardii and E.gentianoides desaturases. Visual inspection of the multiple sequencealignment shown in FIG. 1 suggests that the P. juliae Δ6 desaturasesequence does not contain any introns. This has been observed in Δ6desaturases from Primula and Echium species (Sayanova et al., 2003,Garcia-Maroto et al., 2002).

Example 2 Yeast Transformation and Expression

Constructs pMON83950 (FIG. 3) and pMON67011 (FIG. 2) were introducedinto the host strain Saccharomyces cerevisiae INVSc1 (Invitrogen), whichis auxotrophic for uracil, using the PEG/Li Ac protocol as described inthe Invitrogen manual for pYES2.1/V5-His-TOPO. Transformants wereselected on plates made of SC minimal media minus uracil with 2%glucose. Colonies of transformants were used to inoculate 5 ml of SCminimal media minus uracil and 2% glucose and were grown overnight at30° C. For induction, stationary phase yeast cells were pelleted andre-suspended in SC minimal media minus uracil supplemented with 2%galactose and grown for 3 days at 15° C. When exogenous fatty acids wereprovide to the cultures, 0.01% LA (Δ9, 12-18:2) was added with theemulsifier 0.1% Tergitol. The cultures were grown for 3 days at 15° C.,and subsequently harvested by centrifugation. Cell pellets were washedonce with sterile TE buffer pH 7.5, to remove the media, and lyophilizedfor 24 h. The host strain transformed with the vector containing theLacZ gene was used as a negative control in all studies.

Lipids were extracted from lyophilized yeast pellets by adding 0.1 mLtoluene and incubating over night at room temperature. Extracted lipidswere converted to fatty acid methyl esters (FAMEs) in situ by additionof 0.5 mL 0.6N sodium methoxide in methanol and incubating for 45 min.The FAMEs were extracted by addition of 0.8 mL 10% (w/v) NaCl and 0.15mL of heptane. The heptane layer containing FAMEs was removed and useddirectly for gas chromatography (GC). The FAMEs were identified on aHewlett-Packard 5890 II Plus GC (Hewlett-Packard, Palo Alto, Calif.)equipped with a flame-ionization detector and a capillary column(omegawax 250; 30 m×0.25 mm i.d.×0.25 μm; Supelco, Bellefonte, Pa.). A100:1 split ratio was used for injections. The injector was maintainedat 250° C. and the flame ionization detector was maintained at 270° C.The column temperature was maintained at 180° C. for 1.5 min followinginjection, increased to 240° C. at 40° C./min, and held at 245° C. for3.38 min.

Table 1 shows the fatty acid composition for yeast expressing P. juliaeclones pMON67011 (PjD6D-2), pMON83950 (PjD6D-1) or Mortierella alpina Δ6desaturase, pMON77205. Expected products for Δ6 desaturation of LA andALA were observed for both P. juliae clones (Table 1, GLA and SDA,respectively), demonstrating that the clones contained in pMON67011 andpMON83950 are Δ6 desaturases. The substrate selectivity was determinedby feeding equal quantities of LA and ALA. M. alpina is a filamentousfungus that accumulates high levels of the n−6 fatty acid arachidonicacid and was expected to have a Δ6 desaturase with an n−6 selectivity.Table 2 shows the n−3:n−6 substrate selectivities of the P. juliae andM. alpina Δ6 desaturases. An n−3:n−6 selectivity of ˜0.8 was observedfor the M. alpina Δ6 desaturase. An n−3:n−6 selectivity of ˜1.5-1.9 wasobserved for both P. juliae Δ6 desaturase clones.

TABLE 1 Comparison of fatty acid composition of yeast expressingdifferent Δ6 desaturases Vector Gene FA in medium LA* GLA* ALA* SDA*pMON67011 P. juliae D6D-2 — 2.0 0.0 0.0 0.0 pMON67011 P. juliae D6D-2 —2.5 0.0 0.1 0.0 pMON67011 P. juliae D6D-2 LA 25.7 14.0 0.0 0.0 pMON67011P. juliae D6D-2 LA 28.4 16.8 0.1 0.0 pMON67011 P. juliae D6D-2 ALA 0.30.1 24.4 16.8 pMON67011 P. juliae D6D-2 ALA 0.3 0.1 30.6 19.0 pMON67011P. juliae D6D-2 LA + ALA 22.7 6.0 18.0 8.5 pMON67011 P. juliae D6D-2LA + ALA 24.3 5.8 20.4 8.9 pMON83950 P. juliae D6D-1 — 2.3 0.0 0.3 0.0pMON83950 P. juliae D6D-1 — 2.3 0.0 0.2 0.0 pMON83950 P. juliae D6D-1 LA26.3 15.0 0.0 0.0 pMON83950 P. juliae D6D-1 LA 23.5 16.6 0.0 0.0pMON83950 P. juliae D6D-1 ALA 0.6 0.2 37.3 17.5 pMON83950 P. juliaeD6D-1 ALA 0.7 0.1 33.9 17.4 pMON83950 P. juliae D6D-1 LA + ALA 18.8 4.317.1 9.4 pMON83950 P. juliae D6D-1 LA + ALA 16.9 4.8 15.7 9.8 pMON77205M. alpina D6D — 1.7 0.0 0.2 0.0 pMON77205 M. alpina D6D — 1.0 0.0 0.00.0 pMON77205 M. alpina D6D LA 56.8 6.0 0.0 0.0 pMON77205 M. alpina D6DLA 25.4 4.6 0.2 0.0 pMON77205 M. alpina D6D ALA 0.5 0.0 69.2 2.6pMON77205 M. alpina D6D ALA 0.9 0.0 23.0 5.0 pMON77205 M. alpina D6DLA + ALA 34.8 1.3 39.7 1.1 pMON77205 M. alpina D6D LA + ALA 18.7 2.818.4 2.2 **Reported as a % of the total for all analytes included in theGC-FID chromatogram, including but not shown (16:0, 16:1, 18:0, 20:0,20:1, 20:2, 22:0, 22:1, 22:2

TABLE 2 Comparison of n-3:n-6 substrate selectivities for P. juliae andM. alpina Δ6 desaturases. FA in % conv % conv Ratio Vector Gene mediumGLA* SDA* n-3:n-6** pMON67011 P. juliae LA + ALA 21.0 32.1 1.53 D6D-2pMON67011 P. juliae LA + ALA 19.2 30.3 1.58 D6D-2 pMON83950 P. juliaeLA + ALA 18.7 35.4 1.89 D6D-1 pMON83950 P. juliae LA + ALA 22.2 38.41.73 D6D-1 pMON77205 M. alpina LA + ALA 3.6 2.8 0.78 D6D pMON77205 M.alpina LA + ALA 12.8 10.5 0.82 D6D *The percentage conversion to GLA wascalculated by dividing the value for GLA (Table 1) by the sum of thevalues for LA and GLA (Table 1). The same calculation was made for SDAusing the sum of ALA and SDA (Table 1). **The n-3:n-6 ratio wascalculated by dividing the % conv. SDA by % conv. GLA.

Example 3 Plant Transformation and Expression of Primula juliaeΔ6-Desaturase

The activity of the P. juliae Δ6-desaturase was evaluated in soybean bycombining it with a Δ15-desaturase from either Neurospora crassa(NcD15D), pMON77245 (FIG. 4), or Aspergillus nidulans (AnD15D),pMON77247 (FIG. 5). The vector pMON77245 was constructed in three steps.First P. juliae Δ6-desaturase (PjD6D-2) was placed behind theseed-specific 7S alpha′ promoter by digesting pMON67011 with Sse8387 I,followed by removal of the 3′ overhangs, and Sal I, and then ligatingthe resulting fragment into the EcoRI and filled-in XhoI sites of theexpression vector pMON68527, generating the vector pMON77243. Second,the PjD6D-2 expression cassette was removed from pMON77243 by digestingwith NotI, followed by a fill-in reaction, and then the resultingfragment was ligated into the EcoRV site of the 2T binary vectorpMON77244. Finally, a codon-optimized NcD15D (SEQ ID NO:17) under thecontrol of a 7S alpha seed-specific promoter was combined with thePjD6D-2 by digesting pMON77227 with NotI and then ligating the resultingNcD15D expression cassette fragment into NotI digested pMON77344 to givepMON77245 (FIG. 4). The vector pMON77247 (FIG. 5) was constructed bydigesting vector pMON77242 with Not I and ligating the resultingexpression cassette fragment comprising a codon-optimized AnD15D (SEQ IDNO:18) linked to the 7S alpha promoter into the NotI site of pMON77244.The vectors pMON77245 and pMON77247 were transformed into soybean usingthe method of Martinell et al. (U.S. Pat. No. 6,384,301, the disclosureof which is incorporated herein by reference in the entirety).

Expression of the PjD6D-2 coding sequence was measured by determiningthe fatty acid composition of immature (approximately 30 days afterflowering) R1 transgenic soybean seeds, including both homozygotes andheterozygotes, by gas chromatography of lipid methyl ester derivatives(PCT US03/16144, filed May 21, 2003, the entire disclosure of which isspecifically incorporated herein by reference). The levels of PA(palmitic acid, 16:0), SA (stearic acid, 18:0), OA, LA, GLA, ALA, andSDA are expressed as a percentage of the total weight of measured fattyacids and are shown in Tables 3 and 4 below. The non-transgenic controlline was A3525. Whenever possible, five individual seeds were analyzedfrom each event.

Individual seed from a majority of the pMON77245 transgenic events werefound to accumulate measurable amounts of SDA. In all cases, the levelsof SDA were greater than those of GLA, with an average SDA:GLA ratio foreach event ranging from 2:1 to a high of 8:1. The highest single seedvalue was observed from event GM_A38083, which contained 32.0% SDA and2.6% GLA, with a SDA:GLA ratio of 12:1. Of the 12 events shown below, 9had SDA values >10% in at least one seed out of five. As SDA valuesincreased, the levels of PA, SA and OA did not vary significantly fromcontrol levels; however, there is a strong negative correlation for LA.In seeds that accumulated SDA, the levels of GLA remains low, between2.3 to 5.5%. The ALA levels increased along the SDA levels.

TABLE 3 Relative Area Percent Results (Approx. wt percent) from singlepMON77245-transformed R1 seeds pMON77245 Fatty Acid (percent wt)Pedigree PA SA OA LA GLA ALA SDA A3525 11.47 5.21 16.5 56.75 0 9.15 0A3525 11.66 4.53 18.54 54.9 0 9.51 0 A3525 11.8 5.42 16.66 56.04 0 9.140 A3525 11.41 4.91 17.64 56 0 9.08 0 A3525 11.56 4.36 17.86 56.55 0 8.770 GM_A38005 12.57 4.19 18.45 53.99 0 10.8 0 GM_A38005 13.73 4.77 19.3252.42 0 9.76 0 GM_A38005 14.81 4.74 19.09 36.84 5.23 10.3 8.98 GM_A3800513.4 4.71 18.34 53.26 0 10.29 0 GM_A38005 13.21 4.38 19.97 52.19 0 10.250 GM_A38005 13.08 4.78 17.99 53.56 0 10.59 0 GM_A38013 12.91 4.45 19.7240.8 4.57 9.56 7.99 GM_A38013 12.45 4.38 18.9 55.04 0 9.23 0 GM_A3801313.04 4.68 17.38 40.36 4.66 10.27 9.61 GM_A38013 13.26 4.34 17.14 40.034.6 10.17 10.46 GM_A38013 11.67 4.26 22.5 44.26 3.3 8.95 5.05 GM_A3802112.95 4.33 19.39 53.48 0 9.85 0 GM_A38021 13.07 4.87 18.12 54.1 0 9.84 0GM_A38021 13.14 4.27 22.76 34.62 2.3 13.7 9.2 GM_A38021 12.98 4.08 21.5839.6 1.6 13.7 6.45 GM_A38021 13.21 4.34 17.24 29.03 1.78 19.07 15.31GM_A38043 13.1 4.26 19.58 52.44 0 10.62 0 GM_A38043 13.09 4.3 20.0152.83 0 9.77 0 GM_A38043 14.01 4.35 22.05 29.98 4.39 12.18 13.05GM_A38043 13.32 4.26 19.41 51.85 0 11.16 0 GM_A38043 12.8 4.34 19.81 530 10.05 0 GM_A38048 13.44 5.5 18.01 44.46 2.28 10.7 5.61 GM_A38048 13.434.8 18.57 44.25 2.34 10.93 5.68 GM_A38048 13.14 4.47 18.88 44.97 2.3310.78 5.44 GM_A38048 12.98 4.89 17.79 44.92 2.43 11.23 5.76 GM_A3804813.3 4.56 17.95 35.88 3.41 13.15 11.75 GM_A38060 12.73 4.94 17.37 43.164.01 10.4 7.39 GM_A38060 12.85 5.19 15.27 35.1 5.32 11.88 14.39GM_A38060 12.73 4.99 16.41 43.44 3.95 10.25 8.23 GM_A38060 13.06 5.3416.06 42.75 4.04 10.32 8.43 GM_A38060 12.85 5.25 16.45 42.68 4.01 10.398.36 GM_A38064 13.32 5 18.8 42 3.86 10.16 6.87 GM_A38064 13.07 4.7218.97 42.1 3.59 9.95 7.6 GM_A38064 13.45 4.84 19.7 41.67 3.8 9.92 6.62GM_A38064 12.66 4.61 19.09 43.21 3.52 9.85 7.05 GM_A38064 13.03 4.7319.58 36.38 4.94 11.28 10.06 GM_A38069 12.9 4.71 21.24 41.12 2.64 11.435.97 GM_A38069 12.74 4.76 20.35 51.21 0 10.94 0 GM_A38069 12.93 4.7720.5 51.27 0 10.53 0 GM_A38069 13.18 4.69 18.85 38.76 3.3 12.34 8.87GM_A38069 13.08 4.79 19.16 52.08 0 10.89 0 GM_A38083 13.33 5.28 21.7327.31 2.48 15.28 13.35 GM_A38083 12.8 4.96 16.85 11.52 2.64 18.11 32.02GM_A38083 12.32 5.07 22.23 13.59 2.52 17.46 25.56 GM_A38083 13.22 4.2620.83 15.89 3.81 14.69 26.12 GM_A38083 13.74 4.61 17.03 20.93 4.84 13.8223.91 GM_A38084 12.9 4.04 22.66 41.63 3.37 9.07 5.28 GM_A38084 13.383.94 28.07 25.81 4.9 11.37 11.42 GM_A38084 13.92 3.75 31.36 32.26 2.899.23 5.51 GM_A38084 14.42 4.12 27.17 33.26 3.28 11.57 5.77 GM_A3808412.74 3.95 22.59 40.82 3.3 9.68 5.91 GM_A38089 13.05 4.48 22.37 42.632.55 9.3 4.59 GM_A38089 13.15 4.63 18.82 53.48 0 9.03 0 GM_A38089 12.674.41 20.59 51.87 0 9.42 0.07 GM_A38089 12.64 4.29 20.56 52.58 0 8.96 0GM_A38089 12.72 4.57 21.81 50.79 0 9.16 0 GM_A38094 12.62 4.57 18.9752.96 0 9.9 0.11 GM_A38094 13.3 5.08 17.08 34.49 5.35 11.39 12.35GM_A38094 13.08 4.52 18.38 38.95 5.41 9.88 8.82 GM_A38094 13.41 5 17.2738.5 5.49 10.26 9.1 GM_A38094 12.58 4.46 20.06 40.28 4.88 9.5 7.25

Individual seed from the pMON77247 transgenic events accumulated similaramounts of SDA as compared to pMON77245, with the exception of eventGM_A38083 that accumulated significantly higher levels of SDA. Thelevels of PA, SA, OA, and LA were similar to the control levels shown inTable 3. Generally, the levels of SDA were greater than those of GLAwith an average SDA:GLA ratio for each event ranging from 1:1 to 1.6:1,which was less than that observed for pMON77245.

TABLE 4 Relative Area Percent Results (Approx. wt percent) from singlepMON77247 R1 seeds pMON77247 Fatty Acid (percent wt) Pedigree PA SA OALA GLA ALA SDA GM_A38909 12.18 4.19 20.66 44.94 3.52 8.65 4.87 GM_A3890912.25 3.84 22.37 44.89 2.95 8.22 4.46 GM_A38909 12.06 4.67 22.95 43.373.31 8.32 4.86 GM_A38909 12.64 4.63 17.61 45.99 3.66 9.01 5.44 GM_A3890912.28 4.2 19.42 46.1 3.1 9.01 4.82 GM_A38941 13.95 4.87 18.03 40.2 7.087.87 6.92 GM_A38941 13.76 4.38 19.72 33.62 8.94 8.57 9.95 GM_A3894113.15 4.91 17.89 52.06 0.75 9.52 0.8 GM_A38941 12.73 4.27 22.23 42.144.98 7.44 5.15 GM_A38941 12.73 4.34 19.37 52.34 0.36 9.53 0.37 GM_A3894613.02 4.68 17.4 44.66 4.54 8.83 5.89 GM_A38946 13.17 4.42 17.35 43.715.01 8.91 6.49 GM_A38946 13.63 4.24 18.96 38.16 6.36 8.89 8.75 GM_A3894613.32 4.6 17.76 43.37 4.8 8.94 6.2 GM_A38946 13.32 4.5 18.07 43.24 4.718.95 6.23 GM_A38977 13.43 5.18 21.3 40.54 4.43 8.51 5.62 GM_A38977 13.64.92 21.44 40.95 4.26 8.41 5.42 GM_A38977 13.17 4.23 21.61 38.02 5.458.38 8.07 GM_A38977 13.06 4.97 21.93 37.82 5.75 8.63 6.86 GM_A3897713.33 4.5 22.96 37.43 5.54 8.42 6.76 GM_A39047 13.22 4.21 20.95 31.887.8 9.01 11.72 GM_A39047 13.34 4.47 19.14 31.1 7.54 9.9 13.35 GM_A3904713.79 4.32 18.82 32.97 8.26 9.07 11.68 GM_A39047 13.16 4.38 19.34 29.617.94 9.97 14.44 GM_A39047 12.65 4.25 17.48 50.71 1.49 9.92 2.45

Example 4 Activity of the Primula juliae Δ6-Desaturase in Combinationwith the Neurospora crassa Δ15-Desaturase in Canola

The activity of the Primula juliae Δ6-desaturase in combination withNeurospora crassa Δ15-desaturase was evaluated by transforming canolawith the MON82822 (FIG. 7). pMON82822 contained a native NcD15D (SEQ IDNO:19) as well as PjD6D-2, both inserted into a seed-specific expressioncassette under the control of the napin promoter (PCT US03/16144, thedisclosure of which is specifically incorporated herein by reference).

The pMON82822 vector was constructed by first digesting pMON77214 (PCTUS03/16144) with PmeI and BamHI (filled-in) and ligating the resultingnative NcD15D napin cassette into the EcoRV site of the 2T binary vectorpMON71801 to generate pMON82820. Next, pMON82819 was digested with NotI,the ends were filled in and the resulting PjD6D-2 napin expressioncassette was ligated into the filled-in AscI site of pMON82820 togenerate pMON82822.

A second vector, pMON82821, was also constructed containing thecodon-optimized NcD15D (SEQ ID NO:17) and PjD6D-2 pMON82821 by firstdigesting pMON67011 with SalI and Sse8387I and ligating the resultingPjD6D-2 fragment into the SalI and XhoI (filled-in) sites of the napinexpression cassette in pMON82800 giving pMON82819. The napin cassettecontaining a codon-optimized NcD15D was constructed by digestingpMON67024 with PmeI and BamHI (filled-in) and ligating the resultingfragment into an EcoRV-digested 2T binary vector, pMON71801, givingpMON82801. Finally, pMON82819 was digested with NotI, filled-in and theresulting PjD6D-2 napin expression cassette was ligated into the filledin NotI site of pMON82801 giving pMON82821.

pMON82822 was transformed into canola (Brassica napus) using amodification of the protocol described by Radke et al., (Plant CellReports 11:499-505, 1992). Briefly canola seed of the cultivar ‘Ebony’(Monsanto Canada, Inc., Winnipeg, Canada) were disinfected andgerminated in vitro as described in Radke et al., 1992. Precocultivationwith tobacco feeder plates, explant preparation and inoculation ofexplants with Agrobacterium tumefaciens strain ABI (Koncz and Schell,Mol Gen Genet 204:383-396 (1986)) containing the desired vector were asdescribed with the Agrobacterium being maintained in LB media (solid orliquid) containing 75 mg/l spectinomycin, 25 mg/l chloramphenicol and 50mg/l kanamycin. For plant transformation including callus induction,shoot regeneration, maturation and rooting, glyphosate selection wasused rather than the kanamycin selection as described in Radke et al.,1992. Specifically, the B5-1 callus induction medium was supplementedwith 500 mg/l carbenicillin and 50 mg/l Timentin (Duchefa Biochemie BV)to inhibit the Agrobacterium growth and kanamycin was omitted from themedia. B5BZ shoot regeneration medium contained, in addition, 500 mg/lcarbenicillin, 50 mg/l Timentin and 45 mg/l glyphosate with explantsbeing transferred to fresh medium every two weeks.

Glyphosate selected shoots were transferred to hormone-free B5-0 shootmaturation medium containing 300 mg/l carbenicillin and 45 mg/lglyphosate for two weeks and finally shoots were transferred to B5 rootinduction medium containing 45 mg/l glyphosate. Rooted green plantletswere transplanted to potting soil and acclimated to green houseconditions. Plants were maintained in a greenhouse under standardconditions. The fatty acid composition of mature seed was determined byGC analysis of methyl ester derived lipids as done above for soybeantransformants. The GC analysis of canola seed from plants transformedwith pMON82822 yielded 199 events with SDA levels ranging from 0.12 to4.49% (weight %, 100 seed pool).

Example 5 Construction and Transformation of PjD6D Expression Vectorsfor Soy, Corn and Canola

The expression of the PjD6D sequences alone is evaluated in planta forcanola, corn and soybean under the expression a seed-specific promoter.A soybean expression vector is constructed by digesting pMON77243 withNot I, and ligating the resulting fragment containing PjD6D-2 into theNot I site of the binary vector pMON17227. A canola expression vector isconstructed by digesting pMON83950 with SalI and Sse83871 (made blunt)and ligating the resulting fragment, which contains the coding region ofPjD6D-1 into the SalI and XhoI (filled-in) sites of the seed-specificnapin expression cassette vector pMON82800. The resulting plasmid isthen digested with Not I followed by ligating the resultingnapin-PjD6D-1 expression cassette into the Not I site of the binaryvector pMON17227. A corn expression vector is constructed by digestingpMON83950 with Sal I (filled-in) and Sse83872I (made blunt) and ligatingthe resulting PjD6D-1 fragment into the SfiI (made blunt) site of theglobulin expression cassette in pMON71084. The resulting vector is thendigested with PmeI and HindIII and the expression cassette is thenligated into the HpaI and HindIII sites of the binary vector pMON30167.

The activity of the P. juliae Δ6-desaturase in corn is evaluated incombination with a Neurospora crassa Δ15-desaturase codon-optimized forcorn (NcD15Dnno) (SEQ ID NO:20). The vector pMON67011 is digested withSalI and Sse83871 (made blunt) and the resulting PjD6D-2 fragment isligated into the SfiI (fill-in) site of the globulin expression cassettein pMON71084 to give pMON82823. Next, pMON82806 (PCT US03/16144) isdigested with PmeI and HindIII and the resulting globulin NcD15Dnnocassette is ligated into the NotI (fill-in) and HindIII sites of the 1Tbinary vector pMON30167 to give pMON82824. Finally the globulin PjD6D-2cassette is combined with globulin NcD15Dnno by digesting pMON82823 withPmeI and HindIII and ligating the resulting fragment into the SmaI andHindIII sites of pMON82824 giving pMON82825. The resulting vector isintroduced into maize via Agrobacterium tumefaciens-mediatedtransformation as known to one of skill in the art, e.g., U.S. Pat. No.6,603,061.

Example 6 Cloning of Primula waltonii and Primula alpicola Δ6 DesaturaseSequences

Cloning of the Primula waltonii Δ6 desaturase (PwD6D) and P. alpicola Δ6desaturase (PaD6D) genes was achieved by PCR amplification of a partialinternal genomic DNA region using degenerate oligonucleotides, followedby bi-directional genomic walking. Total genomic DNA was isolated fromP. waltonii and P. alpicola (Collector's Nursery) using the DNeasy PlantMini Kit (Qiagen), following the manufacturer's procedure. Two fragmentswere isolated from the P. alpicola genomic DNA using the degenerateoligonucleotides BO-1 For and BO-2 Rev as described by Garcia-Maroto etal. 2002:

BO-1 For: 5′-ATMAGYATYGGTTGGTGGAARTGG-3′ (SEQ ID NO: 6) BO-2 Rev:5′-AATCCACCRTGRAACCARTCCAT-3′ (SEQ ID NO: 7)

The first P. alpicola fragment was 550 bp in length and corresponded topositions 553 to 1103 of SEQ ID NO:21. This fragment was cloned intopCR®4-TOPO® (Invitrogen) to yield the vector pMON83977 (no intron). Thesecond P. alpicola fragment was 550 bp in length and corresponded topositions 763 to 1313 of SEQ ID NO:23. This fragment was cloned intopCR®4-TOPO® (Invitrogen) to yield the vector pMON83975 (containsintron). One fragment was obtained from P. waltonii that was 550 bp inlength and corresponded to positions 763 to 1313 of SEQ ID NO:25. Thisfragment was cloned into pCR®4-TOPO® (Invitrogen) to yield the vectorpMON83976. The polypeptide sequences encoded by SEQ ID NOs:21, 23 and 25are given in SEQ ID NOs:22, 24 and 26, respectively.

To determine the genomic flanking sequences of the pMON83975, pMON83976,and pMON83977 inserts, a Universal Genome Walker Kit™ (BD Biosciences)was utilized, following the manufacture's procedure. Four genomiclibraries for each Primula species were generated by digesting the DNAwith four restriction enzymes: EcoRV, PvuII, StuI, and DraI. After apurification step, the digestions were ligated to an adapter provided inthe kit. The procedure then involved two PCR reactions, each with agene-specific primer and an adapter-primer. The secondary PCR reactionused a dilution of the primary PCR reaction products as a template.

A. pMON83975 (PaD6D-2)

For the 5′ direction, primers PD6D R7 and PD6D R1 were used for theprimary and secondary PCR reactions, respectively. For the 3′ direction,primers PD6D F7 and PD6D F1 were used for the primary and secondary PCRreactions, respectively. The primer sequences are given below:

PD6D R7: 5′-CACACATGACCGGATAAAACGTCCAGT-3′ (SEQ ID NO: 27) PD6D R1:5′-AGGGATATACTGGAGGTCGGGGTCGTA-3′ (SEQ ID NO: 28) PD6D F7:5′-GAGCTATTCCGTTACGGGGATACAACA-3′ (SEQ ID NO: 29) PD6D F1:5′-TGCAGGGACACTTAACATATCGTGCCC-3′ (SEQ ID NO: 30)

Genome walking in the 5′ direction yielded a 751 bp fragment from theEcoRV library. This product was cloned into pCR®4-TOPO® (Invitrogen)giving pMON83978, and the insert was sequenced. The resulting sequencedid not contain a start codon of the putative delta 6 desaturase geneand thus another set of PCR reactions was performed using gene specificprimers designed to walk in the 5′ direction from the pMON83978 insert.The primers used for the second genome walking set in the 5′ directionwere PD6D R17 and PD6D R16 for the primary and secondary PCR reactions,respectively. The sequences are given below:

PD6D R17: 5′-GTGAAAGTTGTTGAGGAGGGATCGGTA-3′ (SEQ ID NO: 31) PD6D R16:5′-GTGGAAGGAGGATGGTAAGCGAGGAAA-3′ (SEQ ID NO: 32)

A product of 473 bp in length from the PvuII library was cloned intopCR®4-TOPO® giving pMON83980 and the insert was sequenced. This insertcontained a start codon corresponding to position 1 of SEQ ID NO:23.Genome walking in the 3′ direction resulted in a 942 bp fragment fromthe DraI library. This product was cloned into pCR®4-TOPO®, givingpMON83979. The insert was sequenced and found to contain 294 bp of thecoding region for the putative delta 6 desaturase gene, followed by astop codon at position 1549 with respect to SEQ ID NO:23.

The inserts of pMON83975, pMON83978, pMON83980 and pMON83979 werealigned to form a composite sequence of a putative Δ6 desaturase genefor P. alpicola giving PaD6D-2, SEQ ID NO:23. Two primers were designedto PCR amplify the complete open reading frame from P. alpicola genomicDNA. The primer sequences are given below:

Pa D6D F1: 5′- GTCGACATGGCTAACAAATCTCAAACAGGTTAC-3′ (SEQ ID NO: 33) PaD6D R1: 5′- CCTGCAGGTCACCCGAGAGTTTTAACAGCCTCC-3” (SEQ ID NO: 34)The PCR amplified fragment (SEQ ID NO:23) was then ligated into theyeast expression vector pYES2.1-TOPO giving the vector pMON83968.

B. pMON83976 (PwD6D)

A putative Δ6 desaturase was PCR amplified from P. waltonii genomic DNAusing the primers Pa D6D F1 (SEQ ID NO:33) and Pa D6D R1 (SEQ ID NO:34)shown above. The PCR amplified fragment (SEQ ID NO:25) was then ligatedinto the yeast expression vector pYES2.1-TOPO giving the vectorpMON83967.

C. pMON83977 (PaD6D-1)

For the 5′ direction, primers PD6D R9 and PD6D R4 were used for theprimary and secondary PCR reactions, respectively. For the 3′ direction,primers PD6D F9 and PD6D F4 were used for the primary and secondary PCRreactions, respectively. The primer sequences are given below:

PD6D R9: 5′-CACACATTACCGGATAAAACGTCCAGT-3′ (SEQ ID NO: 35) PD6D R4:5′-AGGAATATACTGGAGGTCTGGGTCGTA-3′ (SEQ ID NO: 36) PD6D F9:5′-ATTTTTCTTCGGACGTATACATGGGCC-3′ (SEQ ID NO: 37) PD6D F4:5′-TTCGGGGACACTGAACATATCGTGCCC-3′ (SEQ ID NO: 38)

Genome walking in the 5′ direction yielded a 979 bp fragment from theStuI library. This product was cloned into pCR®4-TOPO® (Invitrogen)giving pMON83981, and the insert was sequenced. The resulting sequencecontained the start codon of the putative delta 6 desaturase at position1 with respect to SEQ ID NO:21. Genome walking in the 3′ directionresulted in a 1028 bp fragment from the DraI library. This product wascloned into pCR®4-TOPO® (Invitrogen), giving pMON83982. The insert wassequenced and found to contain 295 bp of the coding region for theputative delta 6 desaturase gene, followed by a stop codon at position1339 with respect to SEQ ID NO:21.

The inserts of pMON83977, pMON83981 and pMON83982 were aligned to form acomposite sequence of a second putative Δ6 desaturase gene for P.alpicola giving PaD6D-1, SEQ ID NO:21. Two primers were designed to PCRamplify the complete open reading frame from P. alpicola genomic DNA.The primer sequences are given below.

Pf D6D-F2: 5′-GTCGACATGGCCAACACTAGTTACATTTCCAGCT-3′ (SEQ ID NO: 39) PfD6D-R2: 5′-GATATCACCCCAGAGTGTTAACAGCTTCCCAG-3′ (SEQ ID NO: 40)

The PCR amplified fragment was then ligated into the yeast expressionvector pYES2.1-TOPO giving the vector pMON67026 (SEQ ID NO 21).

Alignment of PaD6D-2 and PwD6D (also abbreviated PRIwaD6D) with othercharacterized plant Δ6 desaturase genes revealed that these two genescontained a single intron corresponding to positions 476 to 676 in SEQID NO:23 and positions 476 to 651 in SEQ ID NO:25. This has beenobserved in Δ6 desaturases from Primula and Echium species (Sayanova etal., 2003, Garcia-Maroto et al., 2002).

The three Δ6 desaturase clones encode potential polypeptides of 446amino acids for PaD6D-1 (SEQ ID NO:22), 449 amino acids for PaD6D-2 (SEQID NO: 24) and 449 amino acids for PwD6D (SEQ ID NO: 26). Thesesequences have high similarity to other plant Δ6 desaturases (FIG. 1),including an N-terminal cytochrome b₅ domain, which is found in allfront-end desaturases (Napier et al., 2003). Within the cytochrome b₅domain is found the eight invariant residues characteristic of thecytochrome b₅ superfamily and the H-P-G-G heme-binding motif, which hasbeen shown to be essential for enzymatic activity (Napier et al., 1997,Sayanova et al, 1999, Sperling and Heinz 2001). Within the desaturasedomain of the PaD6D-1, PaD6D-2, and PwD6D desaturases are threeconserved histidine boxes that are characteristic of all membrane-bounddesaturases (Shanklin et al., 1994). A distinguishing feature found inall front-end desaturases is that the third histidine box contains aglutamine residue in the first position (Q-x-x-H-H) instead of ahistidine (Napier et al., 1997, Napier et al., 2003, Sperling and Heinz2001). The deduced amino acid sequence of the PaD6D-1 had approximately80% identity to PaD6D-2 and approximately 80% identity to PwD6D.However, the two intron containing genes, PaD6D-2 and PwD6D, are moresimilar to each other with approximately 97% identity, than the two P.alpicola genes, PaD6D-1 and PaD6D are to each other.

Example 7 Cloning Additional Primula Δ6 Desaturase Sequences

Genomic DNA was isolated from P. farinosa and P. florindae using aSarkosyl/CTAB lysis system. Five grams of tissue from each species wasground in a mortar and pestle with liquid nitrogen until ground into afine powder. The powdered tissue was then resuspended in lysis buffer(140 mM sorbitol, 220 mM Tris-HCl, pH 8.0, 22 mMethylenediaminetetraacetic acid (EDTA), 800 mM sodium chloride (NaCl),1% N-laurylsarcosine and 0.8% hexadecyltrimethyl ammonium bromide(CTAB)) and incubated for 1 hour at 65° C. with gentle inversion every10 minutes. After incubation, 10 ml of chloroform was added to the lysissuspension and incubated at room temperature with gentle rocking for 20minutes. The lysis suspension was centrifuged for 10 minutes at12,000×g. The aqueous layer was transferred to a clean tube and thenucleic acid precipitated with 0.6% isopropanol. The nucleic acid pelletwas resuspended in 4 ml of a solution containing 10 mM Tris-HCl, pH 8.0,1 mM EDTA, 1 M NaCl and 20 mg Proteinase K. The resuspended nucleic acidwas then incubated for 2 hours at 63° C. The Proteinase K was then heatinactivated by incubation at 75° C. for 20 minutes. RNase (2.5 μg) wasadded to the solution and incubated at 37° C. for 1 hour. The solutionwas extracted with an equal volume of phenol:chloroform:isoamylalcohol(25:24:1) 2 times. The purified genomic DNA was then ethanolprecipitated.

Approximately, 3 μg of genomic DNA was digested in separate reactionswith the restriction endonucleases, EcoRI, HindIII, KpnI, SalI and XhoI.After digestion, each reaction was purified using a QIAquick® PCRPurification Kit (Qiagen, Valencia, Calif.), following themanufacturer's protocol. The digested genomic DNA was eluted from thepurification columns using 100 μl of elution buffer supplied in the kit.Ligation favoring intramolecular interactions was performed in a 200 μlvolume using 20 μl of the eluted digested genomic DNA in a PEG-freeligation reaction with 800 units of ligase (M0202L) (New EnglandBiolabs, Beverly Mass.) overnight at 16° C., followed by heatinactivation at 75° C. for 10 minutes. After ligation, the reaction wasagain purified using a QIAquick® PCR Purification Kit. Inverse PCR wasperformed using 6-20 ng of purified ligated DNA and from 10-20 pg ofprimer and the Expand Long Template PCR System (Roche Applied Science,Indianapolis, Ind.). Primers are shown in Table 5 and were designedusing a combination of available sequence data and data covering thedesaturase domain. Thermal cycle conditions consisted of an initialincubation at 94° C. for 2 minutes; 10 cycles of 94° C. for 20 seconds,52° C. for 30 seconds and 68° C. for 8 minutes; followed by 25 cycles of94° C. for 30 seconds, 52° C. for 30 seconds and 68° C. for 8 minutesplus 10 seconds per cycle. After cycling was complete, a furtherincubation at 68° C. for 7 minutes was performed. Inverse PCR libraryproducts visible after agarose electrophoresis were cloned into eitherpCR®2.1-TOPO or pCR®4Blunt-TOPO (Invitrogen) following themanufacturer's protocol. The following inverse library fragments (withapproximate size) were cloned: P. farinosa-EcoRI (6 kb) and P.florindae-HindIII (3 kb). The DNA sequencing was performed on an AppliedBiosystems 3730xl DNA Analyzer, using Big Dye® Terminator v3.0.

TABLE 5 Primers and fragments used in inverse PCR determination of the5′ and 3′ regions of the delta 6 desaturase genes. SEQ ID Species PrimerSequence NO: P. farinosa Pfl 107F1 TGGAGGTCTGGGTCGTAATC 41 Pfl 107R1CTTCGGACGTATACATGGGC 42 P. florindae Pfl 113-1F2 TCGTAATCCAGGCTATTGCA 43Pfl 113-1R2 TTTTCTTCGGACGTCCATGT 44

Putative sequences were aligned with public data to determine theapproximate region of the open reading frame (ORF) covered for eachgene. Primers to amplify the ORF of each gene were designed based uponthe aligned inverse PCR data. Proofreading polymerases were used toamplify the putative delta 6 genes to insure fidelity of the finalproduct. The primers used in the final cloning of the putative delta 6desaturase genes are shown in Table 6. The products were cloned intoeither pUC19 or pCR®4Blunt-TOPO (Invitrogen). DNA sequencing wasperformed on an Applied Biosystems 3730xl DNA Analyzer, using Big Dye®Terminator v3.0. Two putative delta 6 desaturase genes were cloned: P.farinosa (PfaD6D) (pMON84809) (SEQ ID NO:45) and P. florindae (PflD6D)(pMON84810) (SEQ ID NO:47).

TABLE 6 Primers used to amplify delta 6 desaturase genes. SEQ ID PrimerSequence NO: Pfarinosa754F GACGATTTTTGAGTGAGAGTTAATTTGAGTCAATAATA 49Pfarinosa2447R CGACATCATAGACAATCATCAAGACACCGT 50 PflorindaestartFATACCCCCTCAAAACACCCCCAAAT 51 PflorindaestopRCTCAATATCACCCGAGAGTTTTAACAGCCT 52

Two primers were designed to amplify the complete PfaD6D open readingframe from pMON84809. The resulting fragment was ligated into the yeastexpression vector pYES2.1-TOPO giving pMON67065. The two primers aregiven below.

Pfar F1: 5′- GTCGAGAACAATGTCCAACACATATCCACCAAATC -3′ (SEQ ID NO: 53)Pfar R1: 5′- CCTGCAGGTCACCCCAGAGTGTTAACAGCTTC -3′ (SEQ ID NO: 54)

Two primers were designed to amplify the complete PflD6D gene containingtwo exons and one intron from pMON84810. The resulting fragment wasligated into vector pYES2.1-TOPO giving pMON67067. The two primers aregiven below.

Pw F1: 5′- GTCGACATGGCTAACAAATCTCAAAC -3′ (SEQ ID NO: 55) Pw R2: 5′-CCTGCAGGTCACGCGAGAGT -3′ (SEQ ID NO: 56)

The two Δ6 desaturase clones encode potential polypeptides of 454 aminoacids for PfaD6D (SEQ ID NO:46) and 449 amino acids for PflD6D (SEQ IDNO:48). These sequences have high similarity to other plant Δ6desaturases (FIG. 1), including an N-terminal cytochrome b₅ domain,which is found in all front-end desaturases (Napier et al., 2003).Within the cytochrome b₅ domain is found the eight invariant residuescharacteristic of the cytochrome b₅ superfamily and the H-P-G-Gheme-binding motif, which has been shown to be essential for enzymaticactivity (Napier et al., 1997, Sayanova et al, 1999, Sperling and Heinz2001). Within the desaturase domain of the putative PflD6D and PfaD6Ddesaturases are three conserved histidine boxes that are characteristicof all membrane-bound desaturases (Shanklin et al., 1994). Adistinguishing feature found in all front-end desaturases is that thethird histidine box contains a glutamine residue in the first position(Q-x-x-H-H) instead of a histidine (Napier et al., 1997, Napier et al.,2003, Sperling and Heinz 2001).

Example 8 Intron Removal

Alignment of the three Primula clones PaD6D-2 (SEQ ID NO:22), PwD6D (SEQID NO:25), and PflD6D (SEQ ID NO:47) revealed extensive similaritybetween the DNA sequences. PaD6D-2 had approximately 97% identity toPwD6D and approximately 98% identity to PflD6D. PwD6D had approximately98% identity to PflD6D. A 2-step PCR procedure was utilized to removethe intron region from each gene. Briefly, the procedure entails theamplification of the two exons in separate PCRs, followed by a secondround of PCR amplification to combine the two exons together. The sameset of primers was used for each gene amplification because of theextensive similarity between the three Δ6 desaturase genes.

Two sets of primers were designed to amplify exon1 from the PwD6D insertin pMON83967. The size of the amplified product was 475 bp andcorresponded to exon 1 of PwD6D. The two primers are given below.

Pw F1: 5′-GTCGACATGGCTAACAAATCTCAAAC -3′ (SEQ ID NO: 55) Pw R1:5′-GTAATGCCCAGAGTCGTGACCTATCCATCCGCACTGGATCC -3′ (SEQ ID NO: 57)

Exon 2 was PCR amplified from pMON83967 using the primer sequences shownbelow. The size of the amplified product was 875 bp.

Pw F2: 5′- GATCCAGTGCGGATGGATAGGTCACGACTCTGGGCATTACCG -3′ (SEQ ID NO:58)Pw R2: 5′- CCTGCAGGTCACCCGAGAGT-3′ (SEQ ID NO:56)

The amplified exon1 and exon2 products were then combined together withthe primers Pw F1 and Pw R2 to PCR amplify the complete ORF minus theoriginal intron. The resulting 1350 bp fragment was ligated into theyeast expression vector pYES2.1-TOPO giving pMON67062.

The removal of the intron region from PaD6D-2 in pMON83968 and PflD6D inpMON84810 was done utilizing the same procedure as described above forPwD6D. The sizes of the exons were the same as that of PwD6D. Theresulting 1350 bp combined exon fragments were ligated into the yeastexpression vector pYES2.1-TOPO giving pMON67063 for PaD6D-2 andpMON67064 for PflD6D.

Example 9 Yeast Transformation and Expression

Constructs pMON83950 (FIG. 3), pMON67011 (FIG. 2), pMON67026, pMON67062,pMON67064, and pMON67065 were introduced into the uracil auxotrophicSaccharomyces cerevisiae strain INVSc1 (Invitrogen) using the S.cerevisiae EasyComp Transformation Kit (Invitrogen). Transformants wereselected on plates made of SC minimal media minus uracil with 2%glucose. Colonies of transformants were used to inoculate 5 ml of SCminimal media minus uracil and 2% glucose and were grown overnight at30° C. For induction, stationary phase yeast cells were pelleted andre-suspended in SC minimal media minus uracil supplemented with 2%galactose and grown for 1 day at 25° C. followed by 3 days at 15° C.When exogenous fatty acids were provide to the cultures, 0.01% (v/v) LA(Δ9, 12-18:2) and 0.01% ALA (Δ9, 12, 15-18:3) were added with theemulsifier 0.1% (w/v) Tergitol. The cultures were grown 1 day at 25° C.followed by 3 days at 15° C., and subsequently harvested bycentrifugation. Cell pellets were washed once with sterile TE buffer pH7.5, to remove the media, and lyophilized for 24 h. The host straintransformed with the vector containing the LacZ gene was used as anegative control in all studies.

FAMEs were prepared from lyophilized yeast pellets by transmethylationwith 0.5 mL 5% (v/v) H₂SO₄ in methanol containing 0.075 mg/mL2,6-Di-tert-butyl-4-methoxyphenol for 90 min at 90° C. The FAMEs wereextracted by addition of 0.9 mL 10% (w/v) NaCl and 0.3 mL of heptane.The heptane layer containing FAMEs was removed and used directly for GCas described in Example 2.

The results shown in Table 7 demonstrate that P. juliae clones pMON67011and pMON83950, P. alpicola clones pMON67026 and pMON67063, P. waltoniiclone pMON67062, P. florindae clone pMON67064, and P. farinosa clonepMON67065 exhibit Δ6 desaturase activity in a yeast expression system.The data in Table 8 demonstrate that each Primula clone encodes aprotein with selectivity for either n−3 or n−6 substrate fatty acids.

TABLE 7 Δ6 desaturase activity of Primula clones in a yeast expressionsystem. Vector Gene FA in medium LA* GLA* ALA* SDA* LacZ-1 LacZ — 0.00.0 0.0 0.0 LacZ-2 LacZ — 0.0 0.0 0.0 0.0 LacZ-3 LacZ — 0.0 0.0 0.0 0.0LacZ-1 LacZ LA + ALA 23.5 0.0 20.6 0.0 LacZ-2 LacZ LA + ALA 20.3 0.016.6 0.0 LacZ-3 LacZ LA + ALA 29.1 0.0 28.0 0.0 pMON67011 P. juliaeD6D-2 — 0.0 0.0 0.0 0.0 pMON67011 P. juliae D6D-2 — 0.0 0.0 0.0 0.0pMON67011 P. juliae D6D-2 — 0.2 0.0 0.0 0.0 pMON67011 P. juliae D6D-2LA + ALA 18.7 6.5 12.2 8.4 pMON67011 P. juliae D6D-2 LA + ALA 14.7 5.49.6 7.6 pMON67011 P. juliae D6D-2 LA + ALA 18.6 5.1 14.6 8.8 pMON67026P. alpicola D6D1 — 0.0 0.0 0.0 0.0 pMON67026 P. alpicola D6D-1 — 0.0 0.00.0 0.0 pMON67026 P. alpicola D6D-1 — 0.0 0.0 0.0 0.0 pMON67026 P.alpicola D6D-1 LA + ALA 23.0 3.6 21.8 1.5 pMON67026 P. alpicola D6D-1LA + ALA 19.1 3.7 17.9 1.5 pMON67026 P. alpicola D6D-1 LA + ALA 22.6 3.124.1 1.5 pMON83950 P. juliae D6D-1 — 0.0 0.0 0.0 0.0 pMON83950 P. juliaeD6D-1 — 0.0 0.0 0.0 0.0 pMON83950 P. juliae D6D-1 — 0.0 0.0 0.0 0.0pMON83950 P. juliae D6D-1 LA + ALA 21.2 4.0 14.9 6.7 pMON83950 P. juliaeD6D-1 LA + ALA 13.9 4.2 8.8 6.0 pMON83950 P. juliae D6D-1 LA + ALA 21.74.3 16.8 8.3 pMON67062 P. waltonii D6D — 0.0 0.0 0.0 0.0 pMON67062 P.waltonii D6D — 0.0 0.0 0.0 0.0 pMON67062 P. waltonii D6D — 0.0 0.0 0.00.0 pMON67062 P. waltonii D6D LA + ALA 17.5 5.7 12.1 7.1 pMON67062 P.waltonii D6D LA + ALA 12.8 4.8 8.6 6.0 pMON67062 P. waltonii D6D LA +ALA 20.9 5.2 16.8 8.4 pMON67063 P. alpicola D6D-2 — 0.0 0.0 0.0 0.0pMON67063 P. alpicola D6D-2 — 0.0 0.0 0.0 0.0 pMON67063 P. alpicolaD6D-2 — 0.0 0.0 0.0 0.0 pMON67063 P. alpicola D6D-2 LA + ALA 19.9 3.713.4 6.7 pMON67063 P. alpicola D6D-2 LA + ALA 16.0 3.6 9.5 5.6 pMON67063P. alpicola D6D2- LA + ALA 19.8 3.6 14.9 7.8 pMON67064 P. florindae D6D— 0.0 0.0 0.0 0.0 pMON67064 P. florindae D6D — 0.0 0.0 0.0 0.0 pMON67064P. florindae D6D — 0.0 0.0 0.0 0.0 pMON67064 P. florindae D6D LA + ALA17.4 5.6 12.0 6.9 pMON67064 P. florindae D6D LA + ALA 12.8 4.8 8.3 5.9pMON67064 P. florindae D6D LA + ALA 17.1 4.5 14.6 8.3 pMON67065 P.farinosa D6D — 0.0 0.0 0.0 0.0 pMON67065 P. farinosa D6D — 0.0 0.0 0.00.0 pMON67065 P. farinosa D6D — 0.0 0.0 0.0 0.0 pMON67065 P. farinosaD6D LA + ALA 22.1 0.9 19.7 0.3 pMON67065 P. farinosa D6D LA + ALA 28.80.8 27.5 0.2 pMON67065 P. farinosa D6D LA + ALA 21.1 0.8 22.7 0.3*Reported as a % of the total for all analytes included in the GC-FIDchromatogram, including (16:0, 16:1, 18:0, 20:0, 20:1, 20:2, 22:0, 22:1,22:2)

TABLE 8 Comparison of n-3:n-6 substrate selectivities for Primula Δ6desaturases. % conv. % conv Ratio Sample Vector Gene GLA* SDA* n-3:n-6 1LacZ-1 LacZ 0.00 0.00 0.00 2 LacZ-2 LacZ 0.00 0.00 0.00 3 LacZ-3 LacZ0.00 0.00 0.00 4 pMON67011 P. juliae D6D-2 25.75 40.64 1.58 5 pMON67011P. juliae D6D-2 26.98 44.07 1.63 6 pMON67011 P. juliae D6D-2 21.64 37.781.75 7 pMON67026 P. alpicola D6D-1 13.60 6.39 0.47 8 pMON67026 P.alpicola D6D-1 16.06 7.83 0.49 9 pMON67026 P. alpicola D6D-1 12.12 5.830.48 10 pMON83950 P. juliae D6D-1 15.82 31.14 1.97 11 pMON83950 P.juliae D6D-1 23.23 40.72 1.75 12 pMON83950 P. juliae D6D-1 16.58 32.921.99 13 pMON67062 P. waltonii D6D 24.46 36.80 1.50 14 pMON67062 P.waltonii D6D 27.05 41.15 1.52 15 pMON67062 P. waltonii D6D 19.77 33.411.69 16 pMON67063 P. alpicola D6D-2 15.74 33.48 2.13 17 pMON67063 P.alpicola D6D-2 18.53 36.82 1.99 18 pMON67063 P. alpicola D6D-2 15.3734.39 2.24 19 pMON67064 P. florindae D6D 24.34 36.72 1.51 20 pMON67064P. florindae D6D 27.29 41.56 1.52 21 pMON67064 P. florindae D6D 20.9636.13 1.72 22 pMON67065 P. farinosa D6D 4.07 1.25 0.31 23 pMON67065 P.farinosa D6D 2.77 0.79 0.29 24 pMON67065 P. farinosa D6D 3.70 1.09 0.29*The percentage conversion to GLA was calculated by dividing the valuefor GLA (Table 1) by the sum of the values for LA and GLA (Table 1). Thesame calculation was made for SDA using the sum of ALA and SDA (Table1). **The n-3:n-6 ratio was calculated by dividing the % conv. SDA by %conv. GLA.

Example 10 Arabidopsis Cloning, Transformation and Expression

After confirming activity of the Primula Δ6 desaturases in yeast, thegenes were then cloned into pMON73273 (a binary vector containing theconstitutive 35S CaMV promoter) for expression in Arabidopsis thalianato determine activity in planta. PwD6D and PaD6D-2 were cloned withintrons intact. The following vectors were transformed into Arabidopsis:pMON83961 (MaD6D) (FIG. 8), pMON83962 (PjD6D-1) (FIG. 9), pMON83963(PaD6D-2) (FIG. 10), pMON84964 (PjD6D-2) (FIG. 11), pMON84965 (PaD6D-1)(FIG. 12), and pMON83966 (PwD6D) (FIG. 13).

Arabidopsis plants were grown by sowing seeds onto 4 inch potscontaining reverse osmosis water (ROW) saturated MetroMix 200 (TheScotts Company, Columbus, Ohio). The plants were vernalized by placingthe pots in a covered flat, in a growth chamber at 4-7° C., 8 hourslight/day for 4-7 days. The flats were transferred to a growth chamberat 22° C., 55% relative humidity, and 16 hours light/day at an averageintensity of 160-200 μEinstein/s/m². The cover was lifted and slid back1 inch after germination, and then was removed when the true leaves hadformed. The plants were bottom watered, as needed, with ROW until 2-3weeks after germination. Plants were then bottom watered, as needed,with Plantex 15-15-18 solution (Plantex Corporation Ottawa, Canada) at50 ppm N₂. Pots were thinned so that 1 plant remained per pot at 2-3weeks after germination. Once the plants began to bolt, the primaryinflorescence was trimmed to encourage the growth of axillary bolts.

Transgenic Arabidopsis thaliana plants were obtained as described byBent et al., Science, 265:1856-1860, 1994 or Bechtold et al., C. R.Acad. Sci, Life Sciences, 316:1194-1199, 1993. Cultures of Agrobacteriumtumefaciens strain ABI containing one of the transformation vectorspMON69804, pMON69812, or pMON69815 were grown overnight in LB (10%bacto-tryptone, 5% yeast extract, and 10% NaCl with kanamycin (75 mg/L),chloramphenicol (25 mg/L), and spectinomycin (100 mg/L)). The bacterialculture was centrifuged and resuspended in 5% sucrose+0.05% Silwet-77solution. The aerial portions of whole Arabidopsis thaliana Columbiaplants (at about 5-7 weeks of age) were immersed in the resultingsolution for 2-3 seconds. The excess solution was removed by blottingthe plants on paper towels. The dipped plants were placed on their sidein a covered flat and transferred to a growth chamber at 19° C. After 16to 24 hours the dome was removed and the plants were set upright. Whenplants had reached maturity, water was withheld for 2-7 days prior toseed harvest. Harvested seed was passed through a stainless steel meshscreen (40 holes/inch) to remove debris.

The harvested seeds described above were sown onto flats containing ROWsaturated MetroMix 200 (The Scotts Company). The plants were vernalizedand germinated as described above. After true leaves had emerged, theseedlings were sprayed with Roundup to select for transformed plants.

The fatty acid composition of mature seed (R2) was determined by GCanalysis of methyl ester derived lipids as done above for soybean seed.Values for pooled seed from each transgenic event are shown in Table 9.The n−3 or n−6 substrate selectivities that were observed in the yeastassays were confirmed in planta.

TABLE 9 Fatty Acid Analysis of Arabidopsis Seed Gene Pedigree ConstructPA SA OA LA GLA ALA SDA MaD6D At_S54435:@ pMON83961 7.47 3.73 14.1826.31 2.3 17.3 0.72 MaD6D At_S54436:@ pMON83961 7.44 3.91 14.72 25.511.72 18.57 0.44 MaD6D At_S54437:@ pMON83961 7.65 3.72 14.51 28.49 0.3717.97 0 MaD6D At_S54438:@ pMON83961 7.65 3.53 13.55 25.48 2.09 19.180.87 MaD6D At_S54439:@ pMON83961 7.7 3.51 13.69 27.81 1.63 17.07 0.45MaD6D At_S54440:@ pMON83961 7.38 3.55 14.42 25.95 1.6 18.26 0.53 MaD6DAt_S54441:@ pMON83961 7.24 3.54 13.53 24.24 4.4 17.68 1.52 MaD6DAt_S54442:@ pMON83961 7.29 3.6 14.7 25.31 3.58 16.45 0.98 MaD6DAt_S54443:@ pMON83961 7.01 3.61 14.46 27.25 0.44 18.49 0 MaD6DAt_S54444:@ pMON83961 7.68 3.75 14.34 27.89 1.19 17.95 0.05 PjD6D-1At_S54446:@ pMON83962 7.5 3.34 13.52 25.05 2.06 13.81 5.93 PjD6D-1At_S54447:@ pMON83962 7.29 3.15 14.03 26.18 1.64 14 5.25 PjD6D-1At_S54448:@ pMON83962 7.2 3.08 13.37 27.24 0.49 17 2.72 PjD6D-1At_S54449:@ pMON83962 7.24 3.17 14.28 27.52 0.46 16.65 2.44 PjD6D-1At_S54450:@ pMON83962 7.24 3.18 13.38 26.3 1.32 15.16 4.92 PjD6D-1At_S54451:@ pMON83962 7.53 3.04 14.49 28.01 1.8 13.03 4.79 PjD6D-1At_S54452:@ pMON83962 7.59 3.44 13.16 25.54 1.72 13.3 6.69 PjD6D-1At_S54453:@ pMON83962 7.22 3.21 14.05 26.72 1.14 14.35 4.48 PjD6D-1At_S54454:@ pMON83962 6.98 3.23 13.48 25.12 2.27 12.62 6.55 PjD6D-1At_S54455:@ pMON83962 7.34 3.18 14.63 27.07 0.16 18.57 1.1 PjD6D-1At_S54456:@ pMON83962 7.26 3.44 15.8 27.83 0.5 15.81 2.45 PjD6D-1At_S54457:@ pMON83962 7.41 3.11 14.03 27.39 1.92 12.97 4.95 PjD6D-1At_S54458:@ pMON83962 7.2 3.26 13.38 26.18 1.31 14.54 5.1 PjD6D-1At_S54459:@ pMON83962 7.23 3.16 13.25 26.38 1.32 15.07 4.46 PjD6D-1At_S54460:@ pMON83962 7.21 3.19 13.48 26.35 1.32 14.36 5.16 PjD6D-1At_S54461:@ pMON83962 7.18 3.34 13.5 26.64 0.79 15.65 3.96 PjD6D-1At_S54462:@ pMON83962 7.11 3.15 13.88 27.28 1.12 15.02 3.84 PjD6D-1At_S54463:@ pMON83962 7.4 3.19 13.37 26.35 0.61 17.58 2.93 PjD6D-1At_S54464:@ pMON83962 7.57 3.34 13.72 26.12 1.24 15.26 4.69 PaD6D-2At_S54466:@ pMON83963 7.25 3.18 14.44 26.54 1.46 14.44 4.45 PaD6D-2At_S54467:@ pMON83963 7.28 3.07 14.66 27.82 0.31 17.25 1.59 PaD6D-2At_S54468:@ pMON83963 7.34 3.22 15.05 26.37 2.01 13.14 4.86 PaD6D-2At_S54469:@ pMON83963 6.91 2.94 14.35 26.77 1.32 14.33 4.38 PaD6D-2At_S54470:@ pMON83963 7.36 3.26 13.31 27.8 1.36 13.39 4.52 PaD6D-2At_S54471:@ pMON83963 7.14 3.07 14.38 25.73 3.26 11.32 6.18 PaD6D-2At_S54472:@ pMON83963 7.67 3.28 14.01 27.82 0 19.54 0.3 PaD6D-2At_S54473:@ pMON83963 7.48 3.27 13.95 26.26 2.12 13.24 5.57 PaD6D-2At_S54474:@ pMON83963 7.22 3.01 14.95 27.87 1.02 14.5 3.48 PaD6D-2At_S54475:@ pMON83963 7.44 3.07 13.33 26.46 1.58 14.27 5.24 PaD6D-2At_S54476:@ pMON83963 7.35 3.17 14.22 27.48 0.8 15.51 3.25 PaD6D-2At_S54477:@ pMON83963 8.01 2.7 15.85 30.18 0 16.8 0 PaD6D-2 At_S54478:@pMON83963 7.45 3.05 13.47 27.48 0.13 19.53 0.84 PaD6D-2 At_S54479:@pMON83963 7.14 2.99 15.32 27.71 0.24 17.74 0.9 PaD6D-2 At_S54480:@pMON83963 7.37 3.1 14.8 27.87 0.07 18.64 0.45 PaD6D-2 At_S54481:@pMON83963 7.39 3.2 13.49 27.32 0.1 19.9 0.6 PaD6D-2 At_S54482:@pMON83963 7.29 3.1 13.72 27.63 0.25 17.96 1.63 PaD6D-2 At_S54483:@pMON83963 7.04 2.97 15.2 28.08 0 18.71 0.1 PaD6D-2 At_S54484:@ pMON839637.09 2.89 14.89 28.18 0.05 19.73 0 PaD6D-2 At_S54485:@ pMON83963 7.172.93 15.33 27.21 1.52 13.48 4.57 PjD6D-2 At_S54487:@ pMON83964 7.18 3.0614.91 27.66 0.79 15.58 3 PjD6D-2 At_S54488:@ pMON83964 7.36 3.09 14.1327.75 1.34 14.21 4.15 PjD6D-2 At_S54489:@ pMON83964 7.48 2.9 13.86 27.520.6 16.94 2.95 PjD6D-2 At_S54490:@ pMON83964 7.39 3.08 14.12 27.93 0.6316.23 2.88 PjD6D-2 At_S54491:@ pMON83964 7.35 3.05 15.03 28.07 0 19.040.16 PjD6D-2 At_S54492:@ pMON83964 7.59 3.07 14.84 27.99 0 19.18 0.33PjD6D-2 At_S54493:@ pMON83964 7.36 2.97 13.57 28.18 0.68 16.38 2.96PjD6D-2 At_S54494:@ pMON83964 7.39 3.03 13.37 27.5 0.98 15.71 3.96PjD6D-2 At_S54495:@ pMON83964 7.46 2.98 13.59 26.97 1.02 16.11 3.83PjD6D-2 At_S54496:@ pMON83964 7.65 3.02 14.54 27.83 0.35 17.43 1.87PjD6D-2 At_S54497:@ pMON83964 7.62 2.94 13.64 28.44 0.89 15.27 3.61PjD6D-2 At_S54498:@ pMON83964 7.55 3.06 14.06 27.53 1.01 14.89 4.37PjD6D-2 At_S54499:@ pMON83964 7.19 3.12 14.62 26.77 1.55 13.28 5.14PjD6D-2 At_S54500:@ pMON83964 7.42 2.9 13.83 27.84 0.39 17.55 2.3PjD6D-2 At_S54501:@ pMON83964 7.51 3.09 14.23 28.21 0 19.5 0.1 PjD6D-2At_S54502:@ pMON83964 7.41 3 13.56 27.41 0.81 16.36 3.33 PjD6D-2At_S54503:@ pMON83964 7.33 2.95 13.46 26.74 1.09 15.92 4.28 PaD6D-1At_S54505:@ pMON83965 7.24 2.97 14.25 27.24 0.96 19.3 0.21 PaD6D-1At_S54506:@ pMON83965 7.37 3.12 14.25 26.81 1.26 19.08 0.24 PaD6D-1At_S54507:@ pMON83965 7.48 3.03 15.61 26.86 0.52 18.75 0.09 PaD6D-1At_S54508:@ pMON83965 7.61 3.07 13.41 25.28 2.2 19.67 0.51 PaD6D-1At_S54509:@ pMON83965 7.33 3.24 14.21 25.71 2.32 18.64 0.48 PaD6D-1At_S54510:@ pMON83965 7.66 3.09 15.86 24.84 1.1 18.88 0.23 PaD6D-1At_S54511:@ pMON83965 7.55 3.08 15.2 25.25 0.94 19.36 0.21 PaD6D-1At_S54512:@ pMON83965 7.43 3.16 13.51 26 1.37 19.63 0.29 PaD6D-1At_S54513:@ pMON83965 8.11 3.3 14.94 24.33 0.45 20.26 0.12 PaD6D-1At_S54514:@ pMON83965 7.35 3.14 14.18 26.35 1.36 19.27 0.36 PaD6D-1At_S54515:@ pMON83965 7.52 2.95 12.14 26.65 0.63 22.45 0.21 PaD6D-1At_S54516:@ pMON83965 7.86 3.29 15.13 23.72 0.74 20.03 0.21 PaD6D-1At_S54517:@ pMON83965 7.2 3.49 15.25 27.77 0.26 18.14 0 PaD6D-1At_S54518:@ pMON83965 7.17 2.81 15.7 23.13 0.06 20.4 0 PaD6D-1At_S54519:@ pMON83965 6.9 3.07 15.34 26.65 0.14 19.19 0 PaD6D-1At_S54520:@ pMON83965 8.64 3.7 15.97 20.96 0.97 18.39 0.28 PaD6D-1At_S54521:@ pMON83965 7.2 3.19 13.39 26.03 1.63 19.36 0.32 PaD6D-1At_S54522:@ pMON83965 8.77 3.69 15.83 20.94 0 18.92 0 PaD6D-1At_S54523:@ pMON83965 7.43 3.33 14.1 26.94 0.23 19.93 0 PwD6DAt_S54524:@ pMON83966 7.37 3.17 15.27 25.68 2.72 13.22 4.36 PwD6DAt_S54525:@ pMON83966 7.15 3.38 14.38 25.61 2.86 12.9 4.82 PwD6DAt_S54526:@ pMON83966 6.87 3.6 14.68 27.25 0.23 17.54 1.19 PwD6DAt_S54527:@ pMON83966 7.01 3.45 15.06 26.18 1.43 14.72 3.88 PwD6DAt_S54528:@ pMON83966 7.21 3.04 14.6 27.87 0.11 18.52 0.65 PwD6DAt_S54530:@ pMON83966 7.59 3.17 15.34 21.81 0.77 17.64 2.92 PwD6DAt_S54531:@ pMON83966 7.4 3.58 14.39 26.71 0.4 17.68 1.74 PwD6DAt_S54532:@ pMON83966 6.28 3.44 14.76 24.09 2.51 12.87 6.1 PwD6DAt_S54533:@ pMON83966 7.01 3.48 14.15 25.54 2.01 12.98 5.54 PwD6DAt_S54534:@ pMON83966 7.35 3.35 14.6 26.37 2.25 13.61 4.32 PwD6DAt_S54535:@ pMON83966 7.24 3.56 14.59 27.04 0.45 17.02 2.17 PwD6DAt_S54536:@ pMON83966 7.22 3.54 13.14 25.92 1.49 15.35 4.53 PwD6DAt_S54537:@ pMON83966 7.18 3.6 13.51 26.27 1.61 15.03 4.02 PwD6DAt_S54538:@ pMON83966 7.75 3.29 13.57 25.43 2.33 13.96 5.35 PwD6DAt_S54539:@ pMON83966 7.15 3.13 14.86 26.63 0.16 18.99 0.35 PwD6DAt_S54540:@ pMON83966 7.66 3.28 14.22 26.2 0.97 16.45 3.24 PwD6DAt_S54541:@ pMON83966 7.39 2.98 13.83 27.29 0 20.28 0 PwD6D At_S54542:@pMON83966 7.39 3.32 14.71 26.08 1.56 14 4.18 control At_S54543:@pMON26140 6.82 3.04 14.82 25.91 0 20.07 0 control At_S54544:@ pMON261407.49 3.23 13.69 27.33 0 19.77 0 control At_S54545:@ pMON26140 7.32 3.2315.05 27.47 0 18.6 0 control At_S54546:@ pMON26140 7.52 3.3 13.73 27.150 19.86 0 control At_S54547:@ pMON26140 7.44 3.21 14.21 27.43 0 19.36 0control At_S54548:@ pMON26140 7.39 3.25 14.1 27.05 0 19.59 0 controlAt_S54549:@ pMON26140 7.71 3.28 13.61 27.98 0 20 0 control At_S54550:@pMON26140 7.62 3.24 13.58 28.28 0 18.83 0 control At_S54551:@ pMON261407.52 3.18 14.73 27.27 0 19.78 0 control At_S54552:@ pMON26140 7.44 3.2114.95 27.69 0 18.43 0 control At_S54553:@ pMON26140 7.72 3.26 13.74 27.20 19.94 0 control At_S54554:@ pMON26140 7.3 3.11 15.09 27.73 0 18.75 0control At_S54555:@ pMON26140 7.44 2.99 14.51 29.21 0 18.34 0 controlAt_S54556:@ pMON26140 7.52 3.19 15.22 27.24 0 18.92 0 controlAt_S54557:@ pMON26140 7.49 3.07 14.6 28.87 0 18.17 0 control At_S54558:@pMON26140 7.45 3.11 14.72 27.88 0 18.88 0 control At_S54559:@ pMON261407.63 3.26 14.39 27.12 0 19.57 0 control At_S54560:@ pMON26140 7.74 3.1513.17 28.5 0 19.61 0 control At_S54561:@ pMON26140 7.39 3.15 14.34 27.060 19.42 0 control At_S54562:@ pMON26140 7.25 3.12 15.78 27.96 0 17.93 0control At_S54563:@ pMON26140 7.59 3.24 14.32 27.2 0 19.54 0 controlAt_S54564:@ pMON26140 6.73 2.82 16.17 26.66 0 18.63 0 controlAt_S54565:@ pMON26140 7.2 3 15.14 27.78 0 18.66 0 control At_S54566:@pMON26140 7.33 3.16 14.6 27.28 0 19.26 0

Example 10 Canola Transformation and Expression

The vectors pMON83961, pMON83962, pMON83963, and pMON83964 described inExample 9 were also transformed into Canola according to the methods inExample 4. pMON70500 was included as a negative control. The fatty acidcomposition of leaves was determined by GC analysis of methyl esterderived lipids. The data is shown in Table 10. Again the substrateselectivities observed in yeast and Arabidopsis were confirmed.

TABLE 10 Fatty Acid Analysis of Canola Leaf Tissue Event Construct PA SAOA LA GLA ALA SDA BN_G8912 pMON70500 11.64 0.63 0.39 10.68 0 53.2 0BN_G8913 pMON70500 12.31 0.79 0.57 11.93 0 53.87 0 BN_G8914 pMON7050016.59 1.72 2.09 20.81 0 47.72 0 BN_G8915 pMON70500 11.74 0.82 0.27 7.860 58.66 0 BN_G8918 pMON70500 10.14 0.59 0.35 11.18 0 52.94 0 BN_G8919pMON70500 10.47 0.75 0.43 13.63 0 50.63 0 BN_G8925 pMON70500 11.3 0.720.51 13.69 0 50.95 0 BN_G8926 pMON70500 11.61 0.84 0.77 15.8 0 49.08 0BN_G8928 pMON70500 10.93 0.69 0.63 16.22 0 49.41 0 BN_G8929 pMON7050015.53 2.06 2.18 13.04 0 47.53 0 BN_G9007 pMON83961 14.54 1.83 2.23 11.273.3 46.08 1.5 BN_G9008 pMON83961 16.91 2.38 1.41 10.26 3.81 46.21 2.01BN_G9009 pMON83961 17.11 1.86 3.04 16.21 0.48 47.15 0.23 BN_G9011pMON83961 18.45 2.27 3.2 19.45 7.25 37.69 1.95 BN_G9013 pMON83961 17.952.39 2.66 20.5 1.29 44.84 0.37 BN_G9014 pMON83961 16.65 1.94 1.83 124.73 42.26 2.79 BN_G9033 pMON83962 16.89 2.23 1.16 16.35 0 50.45 2.52BN_G9034 pMON83962 15.83 2.16 1.64 15.89 0 50.66 1.11 BN_G9035 pMON8396216.36 3.18 2.74 23 0 40.73 3.14 BN_G9036 pMON83962 17.01 2.65 2.4 21.090.37 41.23 5.12 BN_G9037 pMON83962 16.08 2.64 1.82 17.68 0.17 44.39 3.29BN_G8828 pMON83963 13.18 1.32 2.58 14 0.15 47.07 4.1 BN_G8829 pMON8396311.56 1.34 1.42 12.07 0.66 37.31 7.55 BN_G8830 pMON83963 12.49 1.37 1.3112.24 0.31 41.45 5.87 BN_G9020 pMON83963 16.66 2.54 4.3 23.54 1.42 41.60 BN_G9021 pMON83963 16.72 1.91 2.01 14.58 0 47.55 1.36 BN_G9024pMON83964 18.32 2.63 2.14 25.17 0.63 37.29 5.34 BN_G9025 pMON83964 18.412.42 3.16 26.57 0 39.23 0.51 BN_G9026 pMON83964 12.23 1.53 1.8 15.080.14 42.48 2.99

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The references listed below are incorporated herein by reference to theextent that they supplement, explain, provide a background for, or teachmethodology, techniques, and/or compositions employed herein.

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1-52. (canceled)
 53. A method of increasing the concentration of EPA inhuman or animal tissue comprising the following steps: a. providing asource of SDA; b. providing additional ingredients; c. combining saidsource of SDA with said additional ingredients to form an ediblecomposition; d. feeding the human or animal said edible composition;and, wherein said source of SDA further comprises GLA and ALA, andwherein the ratio of SDA/GLA is at least about 1.3.
 54. The method ofclaim 53 wherein said tissue is animal tissue.
 55. The method of claim54 wherein said animal is a livestock animal.
 56. The method of claim 55wherein said animal is selected from the group consisting of horses,beef cattle, dairy cattle, swine, poultry, sheep, goats, or fish. 57.The method of claim 56 wherein said animal is a poultry animal.
 58. Themethod of claim 57 wherein said animal is a chicken.
 59. The method ofclaim 53 wherein said source of SDA is a vegetable oil.
 60. The methodof claim 59 wherein said source of SDA is a modified soybean oil. 61.The method of claim 53 wherein said source of SDA comprises at leastabout 1% SDA of total fatty acids.
 62. The method of claim 53 whereinsaid ratio of SDA/GLA is at least about 1.5.
 63. The method of claim 62wherein said ratio of SDA/GLA is at least about 2.0.
 64. The method ofclaim 63 wherein said ratio of SDA/GLA is at least about 2.5.
 65. Themethod of claim 64 wherein said ratio of SDA/GLA is at least about 3.0.66. The method of claim 65 wherein said ratio of SDA/GLA is at leastabout 5.0.
 67. The method of claim 53 wherein the ratio of SDA/ALA is atleast about 0.5.
 68. The method of claim 67 wherein said ratio ofSDA/ALA is at least about 1.0.
 69. The method of claim 68 wherein saidratio of SDA/ALA is at least about 1.5.
 70. The method of claim 53wherein said ratio of SDA/ALA is less than about 1.8.
 71. The method ofclaim 53 wherein said SDA comprises SDA in a form selected from thegroup consisting of conjugated forms, esters, amides, prodrugs of thefatty acids, sodium salts, potassium salts, lithium salts,N-alkylpolyhydroxamine salts, N-methyl glucamine, and ethyl esters. 72.The method of claim 53 wherein said edible composition is selected fromthe group consisting of milk, margarine, sausage, frying oils, foodadditives, eggs, beverages, soft drinks, carbonated beverages, ready tomix beverages, infused foods, fruits, vegetables, sauces, condiments,smoothies, salad dressings, fruit juices, syrups, desserts, puddings,gelatin, icings, fillings, baked goods, bread, frozen desserts, icecreams, sherbets, soft frozen products, modified cheeses, soft frozencreams, soft frozen ice creams, yoghurts, soft frozen toppings, dairywhipped toppings, non-dairy whipped toppings, oils, emulsified products,shortening, margarine, mayonnaise, butter, cooking oil, and intermediatemoisture food, rice, nutrient drinks, soymilk, and soup.
 73. The methodof claim 54 wherein said edible composition is selected from the groupconsisting of animal feed, an animal feed supplement, and dog food. 74.A method of increasing the concentration of DPA in human or animaltissue comprising the following steps: a. providing a source of SDA; b.providing additional ingredients; c. combining said source of SDA withsaid additional ingredients to form an edible composition; d. feedingthe human or animal said edible composition; and, wherein said source ofSDA further comprises GLA and ALA, and wherein the ratio of SDA/GLA isat least about 1.3.
 75. The method of claim 74 wherein said tissue isanimal tissue.
 76. The method of claim 75 wherein said animal is alivestock animal.
 77. The method of claim 76 wherein said animal isselected from the group consisting of horses, beef cattle, dairy cattle,swine, poultry, sheep, goats, or fish.
 78. The method of claim 77wherein said animal is a poultry animal.
 79. The method of claim 78wherein said animal is a chicken.
 80. The method of claim 74 whereinsaid source of SDA is a vegetable oil.
 81. The method of claim 80wherein said source of SDA is an endogenous soybean oil.
 82. The methodof claim 74 wherein said source of SDA comprises at least about 1% SDA.83. The method of claim 74 wherein the ratio of SDA/GLA is at leastabout 1.5.
 84. The method of claim 83 wherein said ratio of SDA/GLA isat least about 2.0.
 85. The method of claim 84 wherein said ratio ofSDA/GLA is at least about 2.5.
 86. The method of claim 75 wherein saidratio of SDA/GLA is at least about 3.0.
 87. The method of claim 76wherein said ratio of SDA/GLA is at least about 5.0.
 88. The method ofclaim 74 wherein the ratio of SDA/ALA is at least about 0.5.
 89. Themethod of claim 88 wherein said ratio of SDA/ALA is at least about 1.0.90. The method of claim 89 wherein said ratio of SDA/ALA is at leastabout 1.5.
 91. The method of claim 74 wherein said ratio of SDA/ALA isless than about 1.8.
 92. The method of claim 74 wherein said SDAcomprises SDA in a form selected from the group consisting of conjugatedforms, esters, amides, prodrugs of the fatty acids, sodium salts,potassium salts, lithium salts, N-alkylpolyhydroxamine salts, N-methylglucamine, and ethyl esters.
 93. The method of claim 74 wherein saidedible composition is selected from the group consisting of milk,margarine, sausage, frying oils, food additives, eggs, beverages, softdrinks, carbonated beverages, ready to mix beverages, infused foods,fruits, vegetables, sauces, condiments, salad dressings, fruit juices,syrups, desserts, puddings, gelatin, icings, fillings, baked goods,frozen desserts, ice creams, smoothies, sherbets, soft frozen products,soft frozen creams, modified cheeses, soft frozen ice creams, yoghurts,soft frozen toppings, dairy whipped toppings, non-dairy whippedtoppings, oils, emulsified products, shortening, margarine, mayonnaise,butter, cooking oil, and intermediate moisture food, rice, nutrientdrinks, soymilk, and soup.
 94. The method of claim 75 wherein saidedible composition is selected from the group consisting of animal feed,an animal feed supplement, and dog food.
 95. A foodstuff comprising SDAwherein said foodstuff further comprises SDA, GLA, and ALA having aratio of SDA/GLA of at least about 1.3.
 96. The foodstuff of claim 95wherein said foodstuff further comprises at least about 0.1% SDA, basedon the total fatty acid content of the foodstuff.
 97. The foodstuff ofclaim 95 wherein said SDA/GLA ratio is at least about 1.5.
 98. Thefoodstuff of claim 97 wherein said SDA/GLA ratio is at least about 2.0.99. The foodstuff of claim 98 wherein said SDA/GLA ratio is at leastabout 2.5.
 100. The foodstuff of claim 99 wherein said SDA/GLA ratio isat least about 3.0.
 101. The foodstuff of claim 95 further comprising atleast about 0.1% ALA, based on the total fatty acid content of thefoodstuff.
 102. The foodstuff of claim 101 further comprising a ratio ofSDA/ALA wherein said ratio of SDA/ALA is at least about 0.5.
 103. Thefoodstuff of claim 102 wherein said ratio of SDA/ALA is at least about1.0.
 104. The foodstuff of claim 103 wherein said ratio of SDA/ALA is atleast about 1.5.
 105. The foodstuff of claim 104 wherein said ratio ofSDA/ALA is less than about 1.8.
 106. The foodstuff of claim 95 whereinsaid foodstuff is selected from the group consisting of milk, margarine,sausage, frying oils, food additives, eggs, beverages, soft drinks,carbonated beverages, ready to mix beverages, infused foods, fruits,vegetables, sauces, condiments, salad dressings, smoothies, fruitjuices, syrups, desserts, puddings, gelatin, icings, fillings, bakedgoods, frozen desserts, ice creams, sherbets, soft frozen products, softfrozen creams, modified cheeses, soft frozen ice creams, yoghurts, softfrozen toppings, dairy whipped toppings, non-dairy whipped toppings,oils, emulsified products, shortening, margarine, mayonnaise, butter,cooking oil, and intermediate moisture foods, rice, nutrient drinks,soymilk, soup and dog food.
 107. A method of feeding an animalcomprising the following steps: a. providing a seed oil comprising SDAand GLA; b. providing starting feed ingredients; c. combining said seedoil and said feed ingredients to make a feed; d. feeding said feed to ananimal; and wherein said feed has a ratio of SDA/GLA of at least about1.3.
 108. The method of claim 107 wherein said seed oil is a soybeanseed oil.
 109. The method of claim 108 wherein said soybean seed oilcomprises at least about 1% SDA.
 110. The method of claim 109 whereinsaid soybean seed oil comprises at least about 5% SDA.
 111. The methodof claim 110 wherein said ratio of SDA/GLA is at least about 1.5. 112.The method of claim 111 wherein said ratio of SDA/GLA is at least about2.0.
 113. The method of claim 112 wherein said ratio of SDA/GLA is atleast about 2.5.
 114. The method of claim 113 wherein said ratio ofSDA/GLA is at least about 3.0
 115. The method of claim 114 wherein saidseed oil further comprises ALA.
 116. The method of claim 115 whereinsaid seed oil has a ratio of SDA/ALA of at least about 0.5.
 117. Themethod of claim 116 wherein said ratio of SDA/ALA is at least about 1.0.118. The method of claim 117 wherein said ratio of SDA/ALA is at leastabout 1.5.
 119. The method of claim 118 wherein said ratio of SDA/ALA isless than about 1.8.
 120. The method of claim 108 wherein said feedcomprises soybean seeds.
 121. The seeds of claim 107 wherein said seedsare selected from a group consisting of corn, cotton and canola. 122.The method of claim 120 wherein said feed comprises endogenous soybeanseeds having an SDA concentration greater than about 5% and a GLAconcentration less than about 20%.
 123. The method of claim 122 whereinsaid feed comprises a soybean oil having an SDA concentration greaterthan about 10% SDA and less than about 20% GLA.
 124. The method of claim122 wherein said feed comprises a soybean oil having an SDAconcentration greater than about 15% and a GLA concentration less thanabout 20%.
 125. A feed product for an animal comprising a. starting feedingredients; b. soybean oil; and, wherein said soybean oil comprisingSDA, ALA and GLA; and wherein the ratio of SDA/GLA is at least about 1.3and the ratio of SDA/ALA is at least about 0.5.
 126. The feed product ofclaim 125 wherein said ratio of SDA/GLA is at least about 1.5.
 127. Thefeed product of claim 126 wherein said ratio of SDA/GLA is at leastabout 2.0.
 128. The feed product of claim 127 wherein said ratio ofSDA/GLA is at least about 2.5.
 129. The feed product of claim 128wherein said ratio of SDA/GLA is at least about 3.0.
 130. The feedproduct of claim 125 wherein said ratio of SDA/ALA is at least about1.0.
 131. The feed product of claim 130 wherein said ratio of SDA/ALA isat least about 1.5.
 132. The feed product of claim 125 wherein saidratio of SDA/ALA is less than about 1.8.
 133. The feed product of claim125 wherein said feed product is a livestock feed.
 134. The feed productof claim 133 wherein said feed product is a feed selected from the groupconsisting of horse feed, beef cattle feed, dairy cattle feed, swinefeed, poultry feed, sheep feed, goat feed, or fish feed.
 135. The feedproduct of claim 125 wherein said feed product is a feed supplement.136. The feed product of claim 125 wherein said feed product is anintermediate moisture food.
 137. The feed product of claim 136 whereinsaid feed product is selected from the group consisting of dog food andcat food.
 138. The method of claim 125 wherein said feed product furthercomprises an excipient.
 139. A vegetable oil comprising ALA, SDA, andGLA, wherein the ratio of SDA/GLA in said vegetable oil is at leastabout 1.3.
 140. The vegetable oil of claim 139 wherein said SDA/GLAratio is at least about 1.5.
 141. The vegetable oil of claim 140 whereinsaid SDA/GLA ratio is at least about 2.0.
 142. The vegetable oil ofclaim 141 wherein said SDA/GLA ratio is at least about 2.5.
 143. Thevegetable oil of claim 142 wherein said SDA/GLA ratio is at least about3.0.
 144. The vegetable oil of claim 139 wherein said SDA/ALA ratio isat least about 0.5.
 145. The vegetable oil of claim 144 wherein saidratio of SDA/ALA is at least about 1.0.
 146. The vegetable oil of claim145 wherein said ratio of SDA/ALA is at least about 1.5.
 147. Thevegetable oil of claim 146 wherein said ratio of SDA/ALA is less thanabout 1.8.
 148. The vegetable oil of claim 139 wherein said vegetableoil comprises an endogenous soybean seed oil.
 149. The vegetable oil ofclaim 139 wherein said vegetable oil comprises an SDA content of about5% to about 50% and a gamma-linoleic acid content of less than about20%.
 150. The vegetable oil of claim 139 wherein said vegetable oilfurther comprises an excipient.
 151. A nutraceutical compositioncomprising the vegetable oil of any one of claim
 87. 152. Thenutraceutical composition of claim 151 wherein the composition isintended for human consumption.
 153. The nutraceutical composition ofclaim 152 further comprising an excipient.
 154. A therapeuticcomposition comprising the vegetable oil of any one of claim
 139. 155.The therapeutic composition of claim 154 further comprising anexcipient.
 156. A food or feed comprising the vegetable oil of claim139.
 157. A method of topical application of SDA comprising thefollowing steps: a. providing an oil comprising SDA; b. making a lotionor ointment comprising said SDA; c. topically applying said lotion orointment; and, wherein said oil comprises at least about 0.01% SDA. 158.The method of claim 157 wherein said oil further comprises GLA.
 159. Themethod of claim 158 wherein the ratio of SDA/GLA in said lotion is atleast about 1.3.
 160. The method of claim 159 wherein said ratio ofSDA/GLA is at least about 1.5.
 161. The method of claim 160 wherein saidratio of SDA/GLA is at least about 2.0.
 162. The method of claim 161wherein said ratio of SDA/GLA is at least about 2.5.
 163. The method ofclaim 157 wherein said oil further comprises ALA
 164. The method ofclaim 163 further comprising a ratio of SDA/ALA wherein said ratio ofSDA/ALA is at least about 0.5
 165. The method of claim 164 wherein saidratio of SDA/ALA is at least about 1.0.
 166. The method of claim 165wherein said ratio of SDA/ALA is at least about 1.5.
 167. The method ofclaim 166 wherein said ratio of SDA/ALA is less than about 1.8.
 168. Themethod of claim 157 wherein said topically applying comprises applyingto skin.
 169. The method of claim 157 wherein said lotion furthercomprises an excipient.
 170. The method of claim 157 wherein said SDAcomprises SDA in a form selected from the group consisting of conjugatedforms, esters, amides, prodrugs of the fatty acids, sodium salts,potassium salts, lithium salts, N-alkylpolyhydroxamine salts, N-methylglucamine, and ethyl esters.
 171. A lotion or ointment for topicalapplication comprising an oil wherein said oil comprises stearidonicacid (SDA) and gamma linolenic acid (GLA), and wherein the ratio ofSDA/GLA in said oil is at least about 1.3.
 172. The lotion or ointmentof claim 171 wherein said oil is an endogenous soybean oil.
 173. Thelotion or ointment of claim 171 wherein said oil comprises at leastabout 0.1% SDA.
 174. The lotion or ointment of claim 172 wherein saidratio of SDA/GLA is at least about 1.5.
 175. The lotion or ointment ofclaim 173 wherein said ratio of SDA/GLA is at least about 2.0.
 176. Thelotion or ointment of claim 174 wherein said ratio of SDA/GLA is atleast about 2.5.
 177. The lotion or ointment of claim 175 wherein saidratio of SDA/GLA is at least about 3.0.
 178. The lotion or ointment ofclaim 171 wherein said oil further comprises ALA.
 179. The lotion orointment of claim 178 further comprising a ratio of SDA/ALA wherein saidratio of SDA/ALA is at least about 0.5.
 180. The lotion or ointment ofclaim 179 wherein said ratio of SDA/ALA is at least about 1.0.
 181. Thelotion or ointment of claim 180 wherein said ratio of SDA/ALA is atleast about 1.5.
 182. The lotion or ointment of claim 181 wherein saidratio of SDA/ALA is less than about 1.8.
 183. The lotion or ointment ofclaim 171 wherein said lotion or ointment further comprises anexcipient.
 184. The lotion or ointment of claim 171 wherein said SDAcomprises SDA in a form selected from the group consisting of conjugatedforms, esters, amides, prodrugs of the fatty acids, sodium salts,potassium salts, lithium salts, N-alkylpolyhydroxamine salts, N-methylglucamine, and ethyl esters.
 185. The method of claim 54 wherein saidanimal tissue is that of a crustacean.
 186. The method of claim 55wherein said animal tissue is that of a crustacean.
 187. The method ofclaim 107 wherein said feed comprises endogenous corn seeds having anSDA concentration greater than about 5% and a GLA concentration lessthan about 20%.